Download Report

Journal of Neurochemistry, 2004, 89, 529–536
REVIEW
doi:10.1111/j.1471-4159.2004.02346.x
Nitrotyrosine as a marker for peroxynitrite-induced
neurotoxicity: the beginning or the end of the end
of dopamine neurons?
Donald M. Kuhn,*, ,à Stacey A. Sakowski, Mahdieh Sadidi* and Timothy J.
Geddes*,à
*Department of Psychiatry and Behavioral Neurosciences and Center for Molecular Medicine and
Genetics, Wayne State University School of Medicine
àJohn D. Dingell VA Medical Center, Detroit, Michigan, USA
Abstract
This review examines the involvement of nitrotyrosine as a
marker for peroxynitrite-mediated damage in the dopamine
neuronal system. We propose that the dopamine neuronal
phenotype can influence the cytotoxic signature of peroxynitrite. Dopamine and tetrahydrobiopterin are concentrated in
dopamine neurons, and both are essential for their proper
neurochemical function. It is not well appreciated that dopamine and tetrahydrobiopterin are also powerful blockers of
peroxynitrite-induced tyrosine nitration. What is more, the
reaction of peroxynitrite with either dopamine or
tetrahydrobiopterin forms chemical species (i.e. o-quinones
and pterin radicals, respectively) whose cytotoxic effects may
be manifested far earlier than nitrotyrosine formation in the
course of dopamine neuronal damage. A better understanding
of how the dopamine neuronal phenotype modulates the
effects of reactive nitrogen species could reveal early steps in
drug- and disease-induced damage to the dopamine neuron
and form the basis for rational, protective therapies.
Keywords: dopamine, neurotoxicity, peroxynitrite, quinones,
tetrahydrobiopterin, tyrosine nitration.
J. Neurochem. (2004) 89, 529–536.
Peroxynitrite (ONOO–) is a powerful oxidant and cytotoxic
agent formed by the near-diffusion limited reaction between
nitric oxide (NO) and superoxide (O2–) (Koppenol et al.
1992; Huie and Padmaja 1993). Peroxynitrite can damage
DNA, membrane lipids, and mitochondria, and has been
shown to modify proteins at intrinsic methionine, tryptophan,
and cysteine residues (Ischiropoulos and al-Mehdi 1995;
Beckman and Koppenol 1996). Perhaps the best known
property of ONOO– is its ability to nitrate free tyrosine and
tyrosine residues in proteins (Ischiropoulos et al. 1992;
Souza et al. 1999; Ischiropoulos 2003). Commercial antibodies raised against nitrotyrosine have made facile the
detection of tyrosine-nitration events in tissue under a variety
of conditions of oxidative and nitrosative stress. Although
ONOO– is not the only nitrating species in vivo (Augusto
et al. 2002), it is generally proposed that increases in tyrosine
nitration, whether tyrosine is free or part of a polypeptide
chain, reﬂect the actions of ONOO– (Crow and Beckman
1995; Crow and Ischiropoulos 1996). Findings of increased
nitrotyrosine levels have led to the proposition that ONOO–
plays a causative role in various neurological disorders,
including dementia, ischemia, Parkinson’s disease, and
Alzheimer’s disease (Good et al. 1998; Hensley et al.
1998; Lyras et al. 1998; Torreilles et al. 1999). However,
before an etiological role in neuronal damage can be assigned
to ONOO– via its ability to nitrate tyrosine residues, the
phenotype of the neuron under consideration should be taken
Received July 31, 2003; revised manuscript received November 11,
2003; accepted January 7, 2004.
Address correspondence and reprint requests to Donald M. Kuhn,
Department of Psychiatry and Behavioral Neurosciences, Wayne State
University School of Medicine, 2125 Scott Hall, 540 E. Canﬁeld,
Detroit, MI 48201, USA. E-mail: [email protected]
Abbreviations used: BH4, tetrahydrobiopterin; COX-2, cyclooxygenase-2; DA, dopamine; eGFP, enhanced green ﬂuorescent protein;
hDAT, human dopamine transporter; H2O2, hydrogen peroxide; MDMA,
3,4-methylenedioxymethamphetamine; NO, nitric oxide; NO2, nitrogen
dioxide; ROS, reactive oxygen species; O2 , superoxide radical;
6-OHDA, 6-hydroxydopamine; ONOO), peroxynitrite; SV, synaptic
vesicle; TH, tyrosine hydroxylase.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
529
530 D. M. Kuhn et al.
into account. This applies especially to neurons that use
dopamine (DA) as their neurotransmitter.
While this brief review focuses primarily on ONOO– and
the use of nitrotyrosine as a marker for its neurotoxic actions,
it must be recognized that reactive oxygen species (ROS) and
free radicals such as O2–, hydroxyl radical, and hydrogen
peroxide, to list but a few, have also been implicated as
participants in neural damage. After all, O2– is a primary
reactant in the formation of ONOO–. Our intention is not to
downplay the importance of ROS in drug-induced or diseaserelated neurotoxicity, but limitations in the scope of the
present discussion persuade us to yield to numerous,
excellent reviews that discuss ROS and neurotoxicity in the
detail that is deserved (Butterﬁeld et al. 2003; Dauer and
Przedborski 2003; Jenner 2003; Klein and Ackerman 2003).
Tyrosine nitration and dopamine neuronal damage
Parkinson’s disease and MPTP
Parkinson’s disease is a neurodegenerative disorder that
destroys the DA-utilizing neurons of the nigrostriatal system.
While the cause of Parkinson’s disease is not known, genetic,
environmental, and endogenous neurochemical factors probably interact over the span of a lifetime to damage DA
neurons. Oxidative stress and related mechanisms are high
on the list of endogenous factors that contribute to DA
neuronal loss. ONOO–-mediated nitrosative stress was added
to this list when it was demonstrated that MPTP inactivated
tyrosine hydroxylase (TH) via tyrosine nitration (Ara et al.
1998). MPTP is a synthetic DA neuronal toxin that causes
Parkinson’s disease when ingested by humans and animals
alike (Przedborski et al. 2001a). It was already known that
the actions of MPTP were mediated, in part, by NO and O2–
(Przedborski and Jackson-Lewis 1998), and the ﬁnding of
tyrosine nitration after MPTP intoxication led to the conclusion that its mechanism of action, and by extension, one
process by which neurons are lost to Parkinson’s disease, was
related to ONOO– production. Post-mortem brain samples
from individuals with Parkinson’s disease show elevated
levels of nitrotyrosine (Good et al. 1998). It has also been
shown that a-synuclein is tyrosine nitrated by ONOO–
in vitro (Souza et al. 2000) and after treatment of mice with
MPTP (Przedborski et al. 2001b). The ﬁnding of nitrated
a-synuclein in post-mortem brain from individuals with
Parkinson’s disease bolstered the idea that Lewy body
formation, a cardinal pathohistological sign of Parkinson’s
disease, might be fostered by ONOO–-induced tyrosine
nitration of at least a-synuclein (Giasson et al. 2000).
Nitrotyrosine is generally viewed as a molecular marker
for the actions of ONOO–, yet it is possible that this species
possesses intrinsic toxicity of its own. The idea that
nitrotyrosine could contribute to DA neuronal damage was
supported by the ﬁndings that injections of high concentrations of free 3-nitrotyrosine caused striatal neurodegeneration in vivo (Mihm et al. 2001). The striatal damaging
effects of 3-nitrotyrosine were fundamentally similar to
those of the well-known DA neurotoxin 6-hydroxydopamine (6-OHDA) (Mihm et al. 2001). Thus, it appears possible
that free nitrotyrosine could have a causal role in neurodegenerative conditions, but more studies are needed to
establish that toxic levels of free nitrotyrosine are achieved
in brain when damage occurs and to clarify its mechanism
of action.
Neurotoxic amphetamines
The amphetamine analogs methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) have
received broad notoriety as drugs of abuse and it is
increasingly recognized that human abusers of these drugs
suffer long-term neurochemical and cognitive deﬁcits
(McCann et al. 2000). As is the case for Parkinson’s disease,
a great deal of research into the mechanisms of action of the
neurotoxic amphetamines has focused on oxidative stress
(Cadet 2001; Green et al. 2003; Lyles and Cadet 2003) as a
mechanism most likely to mediate the effects of these drugs.
Studies using transgenic mice and pharmacological approaches have suggested that the neurotoxic amphetamines exert
at least some of their toxicity through the production of NO
and O2– (see Davidson et al. 2001). With this background as
a stimulus, it has also been suggested that ONOO– may play
a role in the damaging effects of these drugs as well. Several
studies have now presented evidence that methamphetamine
causes an increase in the levels of free nitrotyrosine in brain
areas known to be targeted for damage (Imam et al. 2001).
6-OHDA
6-OHDA is a well-known and highly speciﬁc DA neurotoxin. The mechanisms of 6-OHDA toxicity have been related in
part to the chemical instability of the compound itself (i.e.
auto-oxidation) as well as to oxidative stress. A role for
nitrosative stress in 6-OHDA-induced toxicity was suggested
by the demonstration that intrastriatal infusions of 6-OHDA
lead to hydroxylation and nitration of phenylalanine in vivo
(Ferger et al. 2001). In addition, the levels of free nitrotyrosine were increased by 6-OHDA, leading Ferger et al. to
conclude that ONOO– was involved in the neurotoxic effects
of 6-OHDA (Ferger et al. 2001). Treatment of primary
mesencephalic cultures with 6-OHDA causes signiﬁcant
losses of DA neurons, an effect that could be prevented by
EUK-134, a superoxide dismutase/catalase mimetic (Pong
et al. 2000). The 6-OHDA-induced nitration of TH that
preceded DA neuronal loss was prevented by EUK-134 as
well (Pong et al. 2000). The basis for EUK-314 prevention
of protein nitration is not understood, as it is not known to
interact directly with ONOO–.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
The DA neuronal phenotype and nitrotyrosine 531
The DA phenotype prevents tyrosine nitration
It is our thesis, presently, that the phenotype of a neuron
could inﬂuence the chemical properties of ONOO– and other
nitrating species, leading to a change in their ability to nitrate
tyrosines. Therefore, neuronal phenotype, and particularly
that of DA neurons, should be considered when characterizing etiological factors and molecular markers of neurotoxic
processes. DA neurons are obviously characterized by their
selective and high content of DA. Why does this matter when
considering the use of nitrotyrosine as a marker for ONOO–
action, or when searching for the mechanisms by which DA
neurons are damaged by drugs or disease? It matters because
of the ability of DA to react with ONOO–. Rice-Evans and
colleagues have shown that DA and ONOO– react avidly to
form the o-quinone of DA (Kerry and Rice-Evans 1998). In
fact, virtually all catechols are converted to their respective
quinones by ONOO– (Kerry and Rice-Evans 1998). In the
process, the tyrosine (i.e. the free amino acid) nitrating
properties of ONOO– are prevented (Pannala et al. 1997).
We investigated the possibility that the protein nitrating
effects of ONOO– could also be mitigated by DA. TH, the
initial and rate-limiting enzyme in DA biosythesis and a
phenotypical marker for DA neurons, was used as a model
protein for a series of in vitro studies. TH is extensively
tyrosine-nitrated by ONOO– (Kuhn et al. 1999a; BlanchardFillion et al. 2001; Kuhn et al. 2002). This effect of ONOO–
is completely prevented by DA, its precursor DOPA, and its
metabolite DOPAC (Park et al. 2003). Because ONOO–
converts DA to its o-quinone, DA-quinone itself was also
tested and found capable of preventing tyrosine nitration of
TH caused by ONOO– (Park et al. 2003). The concentration
of ONOO– must exceed that of DA or its quinone by a factor
of approximately 5 to overcome the anti-nitrating effects of
DA and cause tyrosine nitration of TH. ONOO– is not the
only tyrosine nitrating species and it has even been argued on
the basis of chemical and kinetic mechanisms that ONOO– is
actually an unlikely tyrosine nitrating species in vivo (Pfeiffer
and Mayer 1998; Pfeiffer et al. 2001). A strong case can be
made for nitrogen dioxide (NO2) as an effective nitrating
species in vivo (Augusto et al. 2002; Espey et al. 2002b), so
it was tested as described above for ONOO–. We found that
NO2 caused extensive tyrosine nitration of TH and this effect
was completely prevented by DA, DOPA, and DOPAC (Park
et al. 2003). Therefore, the ability of DA, its precursor, its
metabolites, and its o-quinone to prevent tyrosine nitration
(at least caused by ONOO– or NO2) appears to be quite
general.
These in vitro studies were extended to a cell culture
model to test the possibility that DA could exert anti-nitrating
effects in intact cells. Our initial attempts to detect nitration
of TH after exposure of PC12 cells to ONOO– or SIN-1, a
compound that can generate ONOO– upon decomposition,
were not successful. Therefore, we turned to a method that
allowed direct, real-time measures of tyrosine nitration in
intact cells. Espey and colleagues originally devised this very
clever and elegant approach by showing that the native
ﬂuorescence of green ﬂuorescent protein (eGFP) could be
quenched by ONOO–- or NO2-induced tyrosine nitration,
and not by other reactants that do not cause tyrosine nitration,
such as NO (Espey et al. 2002a). Considering the possibility
that the high catechol content of PC12 cells could suppress
ONOO–-induced tyrosine nitration, we created stable transformants of HEK-293 cells expressing a TH-eGFP fusion
protein. Both elements of the fusion protein retained their
original functionality upon expression in cells. In addition,
these cells were already expressing the human DA transporter (hDAT) which would be used to transport DA into the cell
interior (Ferrer and Javitch 1998). Exposure of these
engineered cells to NO2, but not ONOO–, reduced eGFP
ﬂuorescence, in agreement with the ﬁndings of Espey et al.
(2002a). The activity of cellular TH was also signiﬁcantly
inhibited by NO2. When the hDAT was used to pre-load cells
with DA, the reduction in ﬂuorescence caused by NO2 (i.e.
tyrosine nitration) was prevented. However, the prevention of
tyrosine nitration by DA is not without expense as TH
activity remains inhibited. We conclude from these studies
that cellular DA can prevent the tyrosine nitrating effects of
reactive nitrogen species (Park et al. 2003).
DA neurons contain high concentrations of tetrahydrobiopterin (BH4) (Levine et al. 1979, 1981). BH4 is the natural
and endogenous co-factor for TH in brain (Thony et al.
2000) and serves a similar role for nitric oxide synthase (Bec
et al. 2000). Milstien and Katusic (1999) showed that BH4 is
targeted for oxidation by ONOO– and, in the process, its
properties as a co-factor for nitric oxide synthase are lost. We
have found recently that BH4 completely prevents the
ONOO–-induced nitration of tyrosine residues in TH (Kuhn
and Geddes 2003). In addition to BH4, a series of dihydro(7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin) and tetrahydro-pterins (6,7-dimethyl-tetrahydropterin,
6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, and tetrahydropterin) were also found to block tyrosine
nitration in TH after exposure of the enzyme to either
ONOO– or NO2. The fully oxidized pterins biopterin and
pterin do not have this anti-nitrating effect. The effects of
BH4 on tyrosine nitration were extended to intact cells
(HEK-293 cells not containing the hDAT as described above)
by showing that the reduced pterin effectively prevented
NO2-induced reductions in eGFP ﬂuorescence. Although
reduced pterins prevent nitration of tyrosines in TH, the
catalytic function of the enzyme remains inhibited, as was
observed with DA (above). We conclude from these studies
that BH4, another important constituent of DA neurons, is
very effective in preventing the tyrosine nitrating effects of
ONOO– and NO2 (Kuhn and Geddes 2003). It was
established recently that ONOO– reacts with BH4 6–10
times faster than with ascorbic acid or low molecular weight
thiols (Kuzkaya et al. 2003). Using endothelial cells to study
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
532 D. M. Kuhn et al.
ONOO–-induced alterations in nitric oxide synthase function,
Kuzkaya et al. (2003) showed that ONOO– uncoupled the
enzyme via its ability to oxidize BH4. Indeed, these results
clearly implicate BH4 as a crucial intracellular target for
ONOO–.
The interaction of ONOO– with DA and BH4, both
important elements of the DA neuronal phenotype, clearly
changes their chemical properties to an extent that they can
no longer function as a neurotransmitter or enzyme co-factor,
respectively. In the process of these reactions, the chemical
properties of ONOO– are changed as well – it loses the
ability to cause the post-translational modiﬁcation of tyrosine
nitration.
Is ONOO– likely to interact with DA or BH4 in vivo?
ONOO– preferentially nitrates hydrophobic, transmembrane
tyrosines as compared with tyrosines in the aqueous phase
(Zhang et al. 2003). Nevertheless, if we assume that ONOO–
does reach the cytoplasm of a DA neuron, for the sake of the
present discussion, it could be argued that DA is concentrated within synaptic vesicles (SV) where it might have a very
low probability of interacting with ONOO–, leaving tyrosine
nitration unimpeded. The de novo synthesis of DA starts in
the cytoplasm where TH converts tyrosine to DOPA. The
cytoplasmic concentration of neuronal DA is not known, but
its concentration in a single synaptic vesicle is approximately
25 mM, and its transient concentration in the synaptic cleft
after release is estimated to be as high as 1.6 mM (Garris
et al. 1994). BH4, while not localized selectively in DA
neurons (but it does appear to be concentrated in monoaminergic neurons), can reach concentrations of approximately
100 lM in DA neurons (Levine et al. 1981). BH4 is
synthesized in and localized to the cytoplasm and, with
DA, could present substantial obstacles for the tyrosine-nitrating properties of ONOO–. MPTP and methamphetamine,
drugs that damage DA neurons, and whose mechanisms of
neurotoxicity have been linked at least in part to ONOO– and
tyrosine nitration, share an extremely important neurochemical property that could inﬂuence the effects of reactive
nitrogen species. Both drugs enter the DA presynaptic
neuron through the DAT where they displace vesicular DA
into the cytoplasm and then, by reverse transport, into the
synaptic cleft (Cubells et al. 1994; Lotharius and O’Malley
2000, 2001). Thus, even though it has been suggested that
MPTP and methamphetamine exert their neurotoxicity
through ONOO–-mediated mechanisms (see above), these
drugs increase the intracellular and extracellular levels of DA
to an extent that could suppress tyrosine nitration, at least
during the acute phases of drug intoxication. Indeed, we have
observed that 6-OHDA, like DA, is very powerful in
preventing ONOO–-induced tyrosine nitration of TH (unpublished observations). Interestingly, 6-OHDA causes hydroxylation and nitration of phenylalanine when administered via
dialysis in concentrations of 1–10 mM over a time course of
60 min (Ferger et al. 2001), conditions that would appear to
require sustained production of high levels of ONOO– to
result in nitration of tyrosine residues in proteins. In
summary, it appears highly likely that ONOO– would
encounter DA and BH4 whether it enters DA neurons from
the outside or is produced from within.
Alternative neurotoxic mechanisms of ONOO– beyond
tyrosine nitration
The DA neuronal phenotype certainly has the opportunity
and the capacity to inﬂuence ONOO–-induced tyrosine
nitration. Do mechanisms exist beyond tyrosine nitration
whereby reactive nitrogen species still exert neurotoxicity?
The non-enzymatic oxidation of DA leads to the formation of
DA quinones (Graham 1978). DA and most catechol species
react avidly with ONOO– and NO2 to form quinones. The
catechol quinones are nucleophilic and can react with free
cysteine or with cysteine residues in proteins. We have
shown that both tryptophan hydroxylase (Kuhn and Arthur
1998, 1999) and TH (Kuhn et al. 1999b; Park et al. 2003)
are inhibited when exposed to catechols in the presence of
reactive nitrogen species, yet neither enzyme is tyrosine
nitrated. Quinones derived from the reaction of ONOO– with
DA or DOPA bind to cysteine residues within tryptophan
hydroxylase or TH, resulting in the formation of redoxcycling quinoproteins (Kuhn and Arthur 1998, 1999).
Redox-cycling species, by accepting and donating electrons,
can provoke downstream reactions that generate various
reactive oxygen species (Paz et al. 1991; Velez-Pardo et al.
1996). DA-quinone-modiﬁed TH can even cause the redoxcycling of iron (Kuhn et al. 1999b). It is well known that
redox modiﬁcations of this transition metal can result in the
generation of ROS (e.g. through Fenton’s chemistry) and is
thought to play a role in neurodegeneration (Berg et al. 2001;
Youdim 2003). Justice and colleagues have established
recently that DA-quinones reduce the function of the DAT
though their ability to modify cysteine 342 (Whitehead et al.
2001). In agreement with these results, we have also found
that quinones formed by reaction of DA with ONOO– modify
DAT at cysteine 342 (Park et al. 2002). Thus, the formation
of quinones through the reaction of DA with ONOO– not
only occurs at the expense of tyrosine nitration, but creates
reactive species downstream of these redox-cycling centers
that could have deleterious effects on DA neurons.
Catechol-quinones, pterin radicals, and DA neuronal
damage
The evidence supporting a role for ONOO– as a participant in
DA neuronal damage is compelling. What is less appreciated
is the possible contribution of catechol-derived quinones or
pterin radicals to the process of DA neurodegeneration.
Intrastriatal injections of neurotoxic levels of DA are
associated with the formation of cysteinyl-DA (Hastings
et al. 1996; Hastings and Berman 1999; LaVoie and Hastings
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
The DA neuronal phenotype and nitrotyrosine 533
1999), a measure of DA-quinone formation. Doses of
methamphetamine that cause damage to DA nerve endings
also increase the striatal levels of cysteinyl-DA (LaVoie and
Hastings 1999). DA-quinones have been shown to diminish
the mitochondrial membrane potential (Berman and Hastings
1999) and lead to apoptosis in DA containing cells (Haque
et al. 2003). A role for DA-quinones in MPTP-induced
neuronal damage was suggested by Teismann et al. (2003)
with the demonstration that MPTP increased the production
of prostaglandin E2 and the rate-limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX-2). Surprisingly,
pharmacological inhibition of COX-2 protected against
MPTP-induced DA damage, not by mitigating inﬂammation
or by preventing tyrosine nitration, but by preventing the
formation of DA–quinones (Teismann et al. 2003). Postmortem brain from individuals with Parkinson’s disease
contains elevated levels of cysteinyl–DA (Spencer et al.
1998), and cerebrospinal ﬂuid from patients with Parkinson’s
disease contains antisera that recognize model proteins that
have been modiﬁed by DA–quinones (Rowe et al. 1998).
The inﬂuence of reduced pterins on the response of DA
neurons to ONOO– is quite complex. On one hand, BH4 can
apparently exert neuroprotective effects. BH4 mediates the
preferential resistance of DA neurons to damage caused by
glutathione depletion (Nakamura et al. 2000) and sepiapterin
(a precursor of BH4) even protects against MPTP-induced
neurotoxicity (Madsen et al. 2003). BH4 also lowers O2–
production by nitric oxide synthase (Rosen et al. 2002) and
scavenges O2– in DA neurons (Nakamura et al. 2001).
Removal of O2– by BH4 from the equation
NO + O2– ﬁ ONOO– would reduce formation of the
product. On the other hand, the reaction of BH4 with
ONOO– can lead to the generation of various pterin radicals,
including the trihydrobiopterin radical, and these species
could have deleterious effects if DA neurons are diminished
in their capacity to reduce them back to BH4 (Kohnen et al.
2001; Patel et al. 2002; Kuzkaya et al. 2003). In either case,
these interactions of BH4 with reactive nitrogen species occur
at the expense of tyrosine nitration.
Is nitrotyrosine formation an early or late occurring event
in the process of DA neuronal damage?
The modiﬁcation of critical cellular proteins or organelles by
ONOO–-induced tyrosine nitration has been proposed as an
early occurring insult that culminates in DA neuronal
degeneration (Ara et al. 1998). Our alternative thesis proposes that the DA phenotype plays a critical role in determining
the manner in which ONOO– exerts its effects in DA
neurons. A summary of the foregoing discussion is depicted
in Fig. 1. This hypothetical DA neuron contains numerous
species that ONOO– could encounter after its production, and
all are known to prevent ONOO–-induced tyrosine nitration.
The reaction of DA with ONOO– results in the formation of
quinones at the expense of tyrosine nitration. DA, its catechol
Fig. 1 The DA neuronal phenotype prevents ONOO–-induced tyrosine nitration. A hypothetical DA neuron is depicted and shows intrinsic
factors that would be encountered by ONOO–, whether synthesized
inside or outside of the DA neuron. Some of these factors are highly
specific for the DA neuronal phenotype (DA, BH4, and 6-OHDA as an
exogenous agent) while others are also found in other neurons as well
(NADH, GSH). Drugs like METH and MPTP (in the form of its active
metabolite MPP+) target DA nerve endings as substrates of the
dopamine transporter (DAT) and displace DA from synaptic vesicles
into the cytoplasm. Each of these factors, individually, has been shown
to block ONOO–-induced tyrosine nitration. The intracellular pool of DA
is replenished through de novo synthesis, uptake through the DAT, or
by drug-induced release from SVs. The intracellular pool of BH4 is also
replenished via de novo synthesis and by recycling of dihydro- forms
back to the tetrahydro- species. Possible downstream products
formed in the reaction of ONOO– with each factor are also indicated.
Each of these factors, individually or collectively, has also been
implicated in conditions that damage DA neurons. In summary,
numerous factors found in DA neurons may predominate and preclude
the ONOO–-induced production of nitrotyrosine.
precursor DOPA, its catechol metabolite DOPAC, and the
quinones of these catechols each block ONOO–-induced
nitration of tyrosine residues in TH (Park et al. 2003). BH4
and its dihydro- and tetrahydro- precursors and products also
react with ONOO–, and the result is a prevention of tyrosine
nitration (Kuhn and Geddes 2003). Catechols and reduced
pterins are not the only species found in DA neurons that
interact with ONOO–. NADH and other reduced nicotinamide dinucleotides react with ONOO– and prevent nitration
of free tyrosine (Kirsch and de Groot 1999) and tyrosine
residues in TH (Kuhn and Geddes 2002). GSH can form
disulﬁde links with protein cysteine residues under
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
534 D. M. Kuhn et al.
conditions of oxidative and nitrosative stress, a post-translational modiﬁcation referred to as S-glutathionylation. It is
now known that TH is modiﬁed by S-glutathionylation,
resulting in an inhibition of catalytic activity (Borges et al.
2002). Furthermore, treatment of TH with ONOO– in the
presence of GSH also results in the inhibition of TH activity
via S-glutathionylation at the expense of tyrosine nitration
(Sadidi et al. unpublished observations).
Taken together, these results provoke an obvious question.
Is ONOO–-induced tyrosine nitration an early event in DA
neuronal degeneration or a late occurring effect made
possible by diminished DA and BH4 content and synthetic
capability? As long as DA neurons can synthesize, store, and
metabolize DA, and as long as they can synthesize, store, and
recycle BH4, ONOO–-mediated tyrosine nitration would
likely be suppressed. In its extreme, our thesis would
conclude that tyrosine nitration is a very late occurring event
in the process of DA neuronal degeneration, made possible
by previous and gradual losses of DA, BH4, and probably
numerous other targets of ONOO– that can intercept tyrosine
nitration (e.g. GSH, NADH).
Our thesis assumes that ONOO– or other tyrosine nitrating
species is produced under conditions (disease- or druginduced) that result in damage to DA neurons, and it attempts
to shift emphasis toward the consideration of DA (and its
quinone) and BH4 (and its radicals) as earlier participants in
the degenerative process, and as potential targets for
therapeutic intervention. However, a role for nitrating species
in neurodegenerative conditions needs further substantiation.
As BH4 is essential for the generation of NO via NOS,
diminishing levels of this pterin seen in normal aging and in
Parkinson’s disease (Lovenberg et al. 1979; Williams et al.
1980) could result in lower levels of NO and ONOO–,
reducing the chances for nitrotyrosine formation. Invocation
of ONOO– (and tyrosine nitration) as a common mediator of
the DA neurotoxicity caused by MPTP, 6-OHDA, and the
neurotoxic amphetamines is inconsistent with several notable
differences among the mechanisms of toxicity associated
with these drugs. For example, MPTP and 6-OHDA destroy
DA nerve endings and neurons, while the toxic actions of the
amphetamines are limited to DA nerve endings. The effects
of 6-OHDA are most often tied to ROS generation and
oxidative stress while MPTP (via MPP+) probably exerts its
damaging effects through inhibition of complex I of the
mitochondrial electron transport chain (Dauer and Przedborski 2003).
DA neuronal degeneration proceeds over a course of
decades in humans, both in normal aging and in disease, so it
is not possible to discern if ﬁndings of tyrosine nitration at
the end stage (i.e. in post-mortem brain) reﬂect the same
situation that existed in the earliest stages of DA neuronal
damage. Blanchard-Fillion et al. (2001) have advocated the
development of therapeutic agents that can prevent formation
of nitrating agents as a means of limiting neuronal injury in
Parkinson’s disease. However, it appears that the brain has
already done this in the form of at least DA and BH4.
Acknowledgements
The research described in this review was supported by the National
Institutes of Drug Abuse (DA10756 and DA014692) and by a VA
Merit Award.
References
Ara J., Przedborski S., Naini A. B., Jackson-Lewis V., Triﬁletti R. R.,
Horwitz J. and Ischiropoulos H. (1998) Inactivation of tyrosine
hydroxylase by nitration following exposure to peroxynitrite and
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Proc. Natl
Acad. Sci. USA 95, 7659–7663.
Augusto O., Bonini M. G., Amanso A. M., Linares E., Santos C. C. and
De Menezes S. L. (2002) Nitrogen dioxide and carbonate radical
anion: two emerging radicals in biology. Free Radic. Biol. Med. 32,
841–859.
Bec N., Gorren A. F. C., Mayer B., Schmidt P. P., Andersson K. K. and
Lange R. (2000) The role of tetrahydrobiopterin in the activation of
oxygen by nitric-oxide synthase. J. Inorg. Biochem. 81, 207–211.
Beckman J. S. and Koppenol W. H. (1996) Nitric oxide, superoxide, and
peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271,
C1424–C1437.
Berg D., Gerlach M., Youdim M. B. H., Double K. L., Zecca L., Riederer P. and Becker G. (2001) Brain iron pathways and their
relevance to Parkinson’s disease. J. Neurochem. 79, 225–236.
Berman S. B. and Hastings T. G. (1999) Dopamine oxidation alters
mitochondrial respiration and induces permeability transition in
brain mitochondria: implications for Parkinson’s disease. J. Neurochem. 73, 1127–1137.
Blanchard-Fillion B., Souza J. M., Friel T. et al. (2001) Nitration and
inactivation of tyrosine hydroxylase by peroxynitrite. J. Biol.
Chem. 276, 46017–46023.
Borges C. R., Geddes T., Watson J. T. and Kuhn D. M. (2002) Dopamine
biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylase inhibition during oxidative stress.
J. Biol. Chem. 277, 48295–48302.
Butterﬁeld D. A., Boyd-Kimball D. and Castegna A. (2003) Proteomics
in Alzheimer’s disease: insights into potential mechanisms of
neurodegeneration. J. Neurochem. 86, 1313–1327.
Cadet J. L. (2001) Molecular neurotoxicological models of Parkinsonism: focus on genetic manipulation of mice. Parkinsonism Relat.
Disord. 8, 85–90.
Crow J. P. and Beckman J. S. (1995) The role of peroxynitrite in nitric
oxide-mediated toxicity. Curr. Top. Microbiol. Immunol. 196, 57–
73.
Crow J. P. and Ischiropoulos H. (1996) Detection and quantitation of
nitrotyrosine residues in proteins: in vivo marker of peroxynitrite.
Meth. Enzymol. 269, 185–194.
Cubells J. F., Rayport S., Rajendran G. and Sulzer D. (1994) Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress.
J. Neurosci. 14, 2260–2271.
Dauer W. and Przedborski S. (2003) Parkinson’s disease: mechanisms
and models. Neuron 39, 889–909.
Davidson C., Gow A. J., Lee T. H. and Ellinwood E. H. (2001) Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms
and relevance to human abuse and treatment. Brain Res. Brain Res.
Rev. 36, 1–22.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
The DA neuronal phenotype and nitrotyrosine 535
Espey M. G., Xavier S., Thomas D. D., Miranda K. M. and Wink D. A.
(2002a) Direct real-time evaluation of nitration with green ﬂuorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. Proc. Natl
Acad. Sci. USA 99, 3481–3486.
Espey M. G., Miranda K. M., Thomas D. D., Xavier S., Citrin D., Vitek
M. P. and Wink D. A. (2002b) A chemical perspective on the
interplay between NO, reactive oxygen species, and reactive
nitrogen oxide species. Ann. NY Acad. Sci. 962, 195–206.
Ferger B., Themann C., Rose S., Halliwell B. and Jenner P. (2001)
6-hydroxydopamine increases the hydroxylation and nitration of
phenylalanine in vivo: implication of peroxynitrite formation.
J. Neurochem. 78, 509–514.
Ferrer J. V. and Javitch J. A. (1998) Cocaine alters the accessibility of
endogenous cysteines in putative extracellular and intracellular
loops of the human dopamine transporter. Proc. Natl Acad. Sci.
USA 95, 9238–9243.
Garris P. A., Ciolkowski E. L., Pastore P. and Wightman R. M. (1994)
Efﬂux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain. J. Neurosci. 14, 6084–6093.
Giasson B. I., Duda J. E., Murray I. V., Chen Q., Souza J. M., Hurtig H.
I., Ischiropoulos H., Trojanowski J. Q. and Lee V. M. (2000)
Oxidative damage linked to neurodegeneration by selective alphasynuclein nitration in synucleinopathy lesions. Science 290, 985–
989.
Good P. F., Hsu A., Werner P., Perl D. P. and Olanow C. W. (1998)
Protein nitration in Parkinson’s disease. J. Neuropathol. Exp.
Neurol. 57, 338–342.
Graham D. G. (1978) Oxidative pathways for catecholamines in the
genesis of neuromelanin and cytotoxic quinones. Mol. Pharmacol.
14, 633–643.
Green A. R., Mechan A. O., Elliott J. M., O’Shea E. and Colado M. I.
(2003) The pharmacology and clinical pharmacology of 3,4methylenedioxymethamphetamine (MDMA, ‘Ecstasy’). Pharm.
Rev. 55, 463–508.
Haque M. E., Asanuma M., Higashi Y., Miyazaki I., Tanaka K. and
Ogawa N. (2003) Apoptosis-inducing neurotoxicity of dopamine
and its metabolites via reactive quinone generation in neuroblastoma cells. Biochim. Biophys. Acta 1619, 39–52.
Hastings T. G. and Berman S. B. (1999) Dopamine-Induced Toxicity
and Quinone Modiﬁcation of Protein: Implications for Parkinson’s Disease, pp. 69–89. F. P. Graham Publishing, Johnson City,
TN.
Hastings T. G., Lewis D. A. and Zigmond M. J. (1996) Role of oxidation
in the neurotoxic effects of intrastriatal dopamine injections. Proc.
Natl Acad. Sci. USA 93, 1956–1961.
Hensley K., Maidt M. L., Yu Z., Sang H., Markesbery W. R. and Floyd
R. A. (1998) Electrochemical analysis of protein nitrotyrosine and
dityrosine in the Alzheimer brain indicates region-speciﬁc accumulation. J. Neurosci. 18, 8126–8132.
Huie R. E. and Padmaja S. (1993) The reaction of NO with superoxide.
Free Radic. Res. Commun. 18, 195–199.
Imam S. Z., el-Yazal J., Newport G. D., Itzhak Y., Cadet J. L., Slikker W.
Jr and Ali S. F. (2001) Methamphetamine-induced dopaminergic
neurotoxicity: role of peroxynitrite and neuroprotective role of
antioxidants and peroxynitrite decomposition catalysts. Ann. NY
Acad. Sci. 939, 366–380.
Ischiropoulos H. (2003) Biological selectivity and functional aspects of
protein tyrosine nitration. Biochem. Biophys. Res. Commun. 305,
776–783.
Ischiropoulos H. and al-Mehdi A. B. (1995) Peroxynitrite-mediated
oxidative protein modiﬁcations. FEBS Lett. 364, 279–282.
Ischiropoulos H., Zhu L., Chen J., Tsai M., Martin J. C., Smith C. D. and
Beckman J. S. (1992) Peroxynitrite-mediated tyrosine nitration
catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298,
431–437.
Jenner P. (2003) Oxidative stress in Parkinson’s disease. Ann. Neurol. 53,
S26–S36.
Kerry N. and Rice-Evans C. (1998) Peroxynitrite oxidises catechols to
o-quinones. FEBS Lett. 437, 167–171.
Kirsch M. and de Groot H. (1999) Reaction of peroxynitrite with
reduced nicotinamide nucleotides, the formation of hydrogen peroxide. J. Biol. Chem. 274, 24664–24670.
Klein J. A. and Ackerman S. L. (2003) Oxidative stress, cell cycle, and
neurodegeneration. J. Clin. Invest. 111, 785–793.
Kohnen S. L., Mouithys-Mickalad A. A., Deby-Dupont G. P., Deby C.
M., Lamy M. L. and Noels A. F. (2001) Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical
pathway. Free Radic. Res. 35, 709–721.
Koppenol W. H., Moreno J. J., Pryor W. A., Ischiropoulos H. and
Beckman J. S. (1992) Peroxynitrite, a cloaked oxidant formed by
nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834–842.
Kuhn D. M. and Arthur R. Jr (1998) Dopamine inactivates tryptophan
hydroxylase and forms a redox-cycling quinoprotein: possible
endogenous toxin to serotonin neurons. J. Neurosci. 18, 7111–
7117.
Kuhn D. M. and Arthur R. E. (1999) L-DOPA-quinone inactivates
tryptophan hydroxylase and converts the enzyme to a redox-cycling quinoprotein. Brain Res. Mol. Brain Res. 73, 78–84.
Kuhn D. M. and Geddes T. J. (2002) Reduced nicotinamide nucleotides
prevent nitration of tyrosine hydroxylase by peroxynitrite. Brain
Res. 933, 85–89.
Kuhn D. M. and Geddes T. J. (2003) Tetrahydrobiopterin prevents
nitration of tyrosine hydroxylase by peroxynitrite and nitrogen
dioxide. Mol. Pharmacol. 64, 946–953.
Kuhn D. M., Aretha C. W. and Geddes T. J. (1999a) Peroxynitrite
inactivation of tyrosine hydroxylase: mediation by sulfhydryl
oxidation, not tyrosine nitration. J. Neurosci. 19, 10289–10294.
Kuhn D. M., Arthur R. E. Jr, Thomas D. M. and Elferink L. A. (1999b)
Tyrosine hydroxylase is inactivated by catechol-quinones and
converted to a redox-cycling quinoprotein: possible relevance to
Parkinson’s disease. J. Neurochem. 73, 1309–1317.
Kuhn D. M., Sadidi M., Lu X., Kriepke C., Geddes T., Borges C. and
Watson J. T. (2002) Peroxynitrite-induced nitration of tyrosine
hydroxylase: identiﬁcation of tyrosines 423, 428, and 432 as sites
of modiﬁcation by MALDI–TOF mass spectrometry and tyrosinescanning mutagenesis. J. Biol. Chem. 277, 14336–14342.
Kuzkaya N., Weissmann N., Harrison D. G. and Dikalov S. (2003)
Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid,
and thiols: implications for uncoupling endothelial nitric-oxide
synthase. J. Biol. Chem. 278, 22546–22554.
LaVoie M. J. and Hastings T. G. (1999) Dopamine quinone formation
and protein modiﬁcation associated with the striatal neurotoxicity
of methamphetamine: evidence against a role for extracellular
dopamine. J. Neurosci. 19, 1484–1491.
Levine R. A., Kuhn D. M. and Lovenberg W. (1979) The regional
distribution of hydroxylase cofactor in rat brain. J. Neurochem. 32,
1575–1578.
Levine R. A., Miller L. P. and Lovenberg W. (1981) Tetrahydrobiopterin
in striatum: localization in dopamine nerve terminals and role in
catecholamine synthesis. Science 214, 919–921.
Lotharius J. and O’Malley K. L. (2000) The parkinsonism-inducing drug
1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J. Biol. Chem. 275, 38581–
38588.
Lotharius J. and O’Malley K. L. (2001) Role of mitochondrial dysfunction and dopamine-dependent oxidative stress in amphetamine-induced toxicity. Ann. Neurol. 49, 79–89.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536
536 D. M. Kuhn et al.
Lovenberg W., Levine R. A., Robinson D. S., Ebert M., Williams A. C.
and Calne D. B. (1979) Hydroxylase cofactor activity in cerebrospinal ﬂuid of normal subjects and patients with Parkinson’s disease. Science 204, 624–626.
Lyles J. and Cadet J. L. (2003) Methylenedioxymethamphetamine
(MDMA, Ecstasy) neurotoxicity: cellular and molecular mechanisms. Brain Res. Rev. 42, 155–168.
Lyras L., Perry R. H., Perry E. K., Ince P. G., Jenner A., Jenner P. and
Halliwell B. (1998) Oxidative damage to proteins, lipids, and DNA
in cortical brain regions from patients with dementia with Lewy
bodies. J. Neurochem. 71, 302–312.
Madsen J. T., Jansen P., Hesslinger C., Meyer M., Zimmer J. and
Gramsbergen J. B. (2003) Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures. J. Neurochem. 85, 214–223.
McCann U. D., Eligulashvili V. and Ricaurte G. A. (2000) (+/-)3,4Methylenedioxymethamphetamine (‘Ecstasy’)-induced serotonin
neurotoxicity: clinical studies. Neuropsychobiology 42, 11–16.
Mihm M. J., Schanbacher B. L., Wallace B. L., Wallace L. J., Uretsky N.
J. and Bauer J. A. (2001) Free 3-nitrotyrosine causes striatal neurodegeneration in vivo. J. Neurosci. 21, RC149.
Milstien S. and Katusic Z. (1999) Oxidation of tetrahydrobiopterin by
peroxynitrite: implications for vascular endothelial function. Biochem. Biophys. Res. Commun. 263, 681–684.
Nakamura K., Wright D. A., Wiatr T., Kowlessur D., Milstien S., Lei X.
G. and Kang U. J. (2000) Preferential resistance of dopaminergic
neurons to the toxicity of glutathione depletion is independent of
cellular glutathione peroxidase and is mediated by tetrahydrobiopterin. J. Neurochem. 74, 2305–2314.
Nakamura K., Bindokas V. P., Kowlessur D., Elas M., Milstien S., Marks
J. D., Halpern H. J. and Kang U. J. (2001) Tetrahydrobiopterin
scavenges superoxide in dopaminergic neurons. J. Biol. Chem.
276, 34402–34407.
Pannala A. S., Rice-Evans C. A., Halliwell B. and Singh S. (1997)
Inhibition of peroxynitrite-mediated tyrosine nitration by catechin
polyphenols. Biochem. Biophys. Res. Commun. 232, 164–168.
Park S. U., Ferrer J. V., Javitch J. A. and Kuhn D. M. (2002) Peroxynitrite inactivates the human dopamine transporter by modiﬁcation
of cysteine 342: potential mechanism of neurotoxicity in dopamine
neurons. J. Neurosci. 22, 4399–4405.
Park S., Geddes T. J., Javitch J. A. and Kuhn D. M. (2003) Dopamine
prevents nitration of tyrosine hydroxylase by peroxynitrite and
nitrogen dioxide: is nitrotyrosine formation an early step in dopamine neuronal damage? J. Biol. Chem. 278, 28736–28742.
Patel K. B., Stratford M. R., Wardman P. and Everett S. A. (2002)
Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. Free Radic.
Biol. Med. 32, 203–211.
Paz M. A., Fluckiger R., Boak A., Kagan H. M. and Gallop P. M. (1991)
Speciﬁc detection of quinoproteins by redox-cycling staining.
J. Biol. Chem. 266, 689–692.
Pfeiffer S., Lass A., Schmidt K. and Mayer B. (2001) Protein tyrosine
nitration in mouse peritoneal macrophages activated in vitro and
in vivo: evidence against an essential role of peroxynitrite. FASEB
J. 15, 2355–2364.
Pfeiffer S. and Mayer B. (1998) Lack of tyrosine nitration by peroxynitrite generated at physiological pH. J. Biol. Chem. 273, 27280–
27285.
Pong K., Doctrow S. R. and Baudry M. (2000) Prevention of 1-methyl4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of
tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide
dismutase and catalase mimetic, in cultured dopaminergic neurons.
Brain Res. 881, 182–189.
Przedborski S. and Jackson-Lewis V. (1998) Mechanisms of MPTP
toxicity. Mov. Disord. 13, 35–38.
Przedborski S., Jackson-Lewis V., Naini A. B., Jakowec M., Petzinger
G., Miller R. and Akram M. (2001a) The parkinsonian toxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a technical
review of its utility and safety. J. Neurochem. 76, 1265–1274.
Przedborski S., Chen Q., Vila M., Giasson B. I., Djaldatti R., Vukosavic
S., Souza J. M., Jackson-Lewis V., Lee V. M. and Ischiropoulos H.
(2001b) Oxidative post-translational modiﬁcations of a-synuclein
in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
mouse model of Parkinson’s disease. J. Neurochem. 76, 637–640.
Rosen G. M., Tsai P., Weaver J., Porasuphatana S., Roman L. J., Starkov
A. A., Fiskum G. and Pou S. (2002) The role of tetrahydrobiopterin
in the regulation of neuronal nitric-oxide synthase-generated
superoxide. J. Biol. Chem. 277, 40275–40280.
Rowe D. B., Le W., Smith R. G. and Appel S. H. (1998) Antibodies from
patients with Parkinson’s disease react with protein modiﬁed by
dopamine oxidation. J. Neurosci. Res. 53, 551–558.
Souza J. M., Daikhin E., Yudkoff M., Raman C. S. and Ischiropoulos H.
(1999) Factors determining the selectivity of protein tyrosine
nitration. Arch. Biochem. Biophys. 371, 169–178.
Souza J. M., Giasson B. I., Chen Q., Lee V. M. and Ischiropoulos H.
(2000) Dityrosine cross-linking promotes formation of stable
a-synuclein polymers. Implication of nitrative and oxidative stress
in the pathogenesis of neurodegenerative synucleinopathies.
J. Biol. Chem. 275, 18344–18349.
Spencer J. P., Jenner P., Daniel S. E., Lees A. J., Marsden D. C. and
Halliwell B. (1998) Conjugates of catecholamines with cysteine
and GSH in Parkinson’s disease: possible mechanisms of formation
involving reactive oxygen species. J. Neurochem. 71, 2112–2122.
Teismann P., Tieu K., Choi D. K., Wu D. C., Naini A., Hunot S., Vila M.,
Jackson-Lewis V. and Przedborski S. (2003) Cyclooxygenase-2 is
instrumental in Parkinson’s disease neurodegeneration. Proc. Natl
Acad. Sci. USA 100, 5473–5478.
Thony B., Auerbach G. and Blau N. (2000) Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem. J. 347, 1–16.
Torreilles F., Salman-Tabcheh S., Guerin M. and Torreilles J. (1999)
Neurodegenerative disorders: the role of peroxynitrite. Brain Res.
Brain Res. Rev. 30, 153–163.
Velez-Pardo C., Jimenez Del Rio M., Ebinger G. and Vauquelin G.
(1996) Redox cycling activity of monoamine-serotonin binding
protein conjugates. Biochem. Pharmacol. 51, 1521–1525.
Whitehead R. E., Ferrer J. V., Javitch J. A. and Justice J. B. (2001)
Reaction of oxidized dopamine with endogenous cysteine residues
in the human dopamine transporter. J. Neurochem. 76, 1242–1251.
Williams A., Ballenger J., Levine R., Lovenberg W. and Calne D. (1980)
Aging and CSF hydroxylase cofactor. Neurology 30, 1244–1246.
Youdim M. B. (2003) What have we learned from cDNA microarray
gene expression studies about the role of iron in MPTP-induced
neurodegeneration and Parkinson’s disease? J. Neural Trans.
Suppl. 65, 73–88.
Zhang H., Bhargava K., Keszler A., Feix J., Hogg N., Joseph J. and
Kalyanaraman B. (2003) Transmembrane nitration of hydrophobic
tyrosyl peptides. localization, characterization, mechanisms of
nitration, and biological implications. J. Biol. Chem. 278, 8969–
8978.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 529–536