The role of dopamine as a vulnerability factor and a toxic agent in Parkinson’s disease (PD) is still controversial, yet the presumed
dopamine toxicity is partly responsible for the “DOPA-sparing” clinical practice that avoids using L-3,4-dihydroxyphenylalanine (L-
tion. In contrast, reducing dopamine input to striatum by lesioning the medial forebrain bundle attenuated motor dysfunction. These
dopamine replacement therapy is the mainstay of PD treatment. In addition, our model provides an efficient in vivo approach to test
rons in Parkinson’s disease (PD) remains unknown. Dopamine
2003). This presumed dopamine toxicity is responsible for the
“DOPA-sparing” clinical practice that avoids using
dihydroxyphenylalanine (L-DOPA), a dopamine precursor, in
early PD. However, there is a lack of in vivo studies on animal
models that directly isolate dopamine as the determining factor.
Dopamine is a very reactive molecule compared with other
neurotransmitters, and dopamine degradation naturally pro-
duces oxidative species (Graham, 1978). More than 90% of do-
pamine in dopamine neurons is stored in abundant terminal
vesicles and is protected from degradation (Eisenhofer et al.,
2004). However, a small fraction of dopamine is cytosolic, and it
is the major source of dopamine metabolism and presumed tox-
icity. Cytosolic dopamine undergoes degradation to form 3,4-
dihydroxyphenylacetic acid (DOPAC) and homovanillic acid
(HVA) as well as hydrogen peroxide via the monoamine oxidase
mine undergoes oxidation to form superoxide, hydrogen perox-
or proteins to form cysteinyl-dopamine and cysteinyl-DOPAC
conjugates (Hastings and Zigmond, 1994; Sulzer and Zecca,
2000; Barzilai et al., 2003).
cell death (Walkinshaw and Waters, 1995; Simantov et al., 1996;
Koshimura et al., 2000). In vivo, one of us demonstrated that a
dopamine terminal loss (Hastings et al., 1996). Sustained eleva-
tion of extracellular dopamine causes striatal neuron degenera-
tion in the dopamine transporter (DAT) knock-out mice (Cyr et
Ministry of Science and Technology, Republic of Korea (U.J.K.); National Institute of Neurological Disorders and
Burke for their help with neuropathology; William Dauer for critical reading of this manuscript; and Nancy Tian,
Jung Kang, Department of Neurology, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637,
TheJournalofNeuroscience,January9,2008 • 28(2):425–433 • 425
al., 2003). Both studies suggest that extracellular dopamine is
toxic while leaving the role of cytosolic dopamine on neurode-
recent studies examined mice with severely reduced vesicular
striatal dopamine and motor performance in the absence of ni-
gral cell loss. Using the same transgenic line, Caudle et al. (2007)
reported mild substantia nigra dopamine neuron loss in aged
mice, suggesting that cytosolic dopamine-mediated toxicity
for neurodegeneration is lacking.
In the present study, we have generated transgenic mice in
which striatal neurons are engineered to take up extracellular
rons. These mice develop motor dysfunctions and progressive
neurodegeneration in the striatum within weeks. Therefore, we
idence that chronic exposure to unregulated cytosolic dopamine
alone is sufficient to produce neurodegeneration in vivo.
Mice. For most experiments, mice were kept on dietary doxycycline
tion. Two- to four-month-old CaMKII?-tTA; tetO-DAT double trans-
genic mice (CamDAT mice) were used after DOX withdrawal. When
CamDAT mice maintained on DOX treatment were used as controls,
sex- and age-matched littermates were used. When tetO-DAT single
transgenic mice (TetDAT mice) were used as controls, hemizygous
water. Behavioral tests were performed during the light period. All ani-
mal procedures were approved by the Institutional Animal Care and
Usage Committee of The University of Chicago.
In situ hybridization and immunohistochemistry. Mice were perfused
lipore, Billerica, MA). For GFAP and ?-synuclein immunostaining, sec-
tions from the same brains were incubated with rabbit anti-GFAP pri-
anti-?-synuclein primary antibody (1:2000, BD Transduction, Lexing-
ton, KY). Sections were then incubated with secondary antibody (Jack-
son ImmunoResearch, West Grove, PA) and ABC (Vector Labs, Burlin-
game, CA). DAB was used for visualization.
Frozen sections (20 ?m) were used for in situ hybridization protocol.
In situ probe for mouse DAT corresponds to the sequence from 1053 to
1801 nt (GenBank accession number NM_010020). In situ probes for
mouse preprodynorphin (Pdyn) and mouse preproenkephalin (PPE)
correspond to the sequence from 120 to 614 nt (GenBank accession
number U64968) and the sequence from 342 to 1025 nt (GenBank ac-
cession number M13227), respectively. Digoxigenin nonradioactive in
expression (Ding et al., 2007).
Western blot. For quantification of DAT protein expression levels,
brains were lysed in NaCl-Tris-EDTA buffer, and 50 ?g of protein was
separated on SDS-polyacrylamide gel. The membrane was then blocked
and incubated with rat anti-mouse DAT antibody (1:2000, Millipore)
and mouse anti-mouse glyceraldehyde-3-phosphate dehydrogenase (1:
200,000, Abcam, Cambridge, MA) sequentially. Signals were then de-
tected by horseradish peroxidase-conjugated secondary antibodies
(Jackson ImmunoResearch) and enhanced chemiluminescence (Fisher,
Hanover Park, IL).
Unbiased stereological cell counting and brain volume estimation. Mice
?m serial sections. One of six sections were stained with mouse anti-
300 ?m grid and 18 ? 18 ?m counting frame were used. The Gunder-
sun’s coefficient of error was 0.05 (m ? 1) in this study. To quantify
sections was used. The detailed method was described in our previous
publication (Chen et al., 2005).
Slides were scanned, and the areas of cerebral cortex, striatum, and
olfactory bulb were measured using ImageJ (NIH). Volumes of these
structures were estimated by Cavalier principles.
3 for no DOX group) were perfused with 4% paraformaldehyde and 2%
Then blocks were processed by osmium tetroxide, dehydrated, and em-
bedded in epoxy resin. Ninety nanometer sections were cut and exam-
ined under Tecnai F30 scanning transmission electron microscope.
HPLC. Tissue dopamine, metabolites, and protein cysteinyl-catechols
were measured according to previously published methods (LaVoie and
Hastings, 1999). Briefly, tissues were homogenized in 1 ml of cold 0.1N
perchloric acid and centrifuged (30,000 ? g, 20 min), and the resulting
supernatant was separated from the protein pellet. An aliquot of the
supernatant was filtered before analysis for dopamine, DOPAC, and
HVA levels on HPLC with electrochemical detection. The precipitated
protein pellet was hydrolyzed in 6N HCl (100°C, under vacuum, 24 h),
ical detection for cysteinyl-catechol conjugates. All catechols were iden-
tified and quantified by comparison to standards.
Open field locomotor activity and food consumption. Each mouse was
Med Associates, Georgia, VT). Illumination of open filed was set to 20
preceding 10 min habituation period.
For home cage food consumption, mice were individually housed.
chow was recorded after 24 h. For small pellet consumption, mice were
tested weekly in transparent cylinder with small pellets (20 mg of each)
for 5 min. The number of pellets consumed was recorded.
Unilateral lesion of dopamine neurons and forepaw adjust stepping test.
Mice were anesthetized and put into stereotaxics. Desipramine (25 mg/
kg, Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally to pro-
3 mg/ml dissolved in 0.01% ascorbate in saline, Sigma-Aldrich) was in-
jected into left medial forebrain bundle (coordinates: anteroposterior,
?1.3 mm from bregma; lateral, 1.3 mm from bregma; dorsoventral,
?4.9 to ?5.2 mm from the skull surface) via a 28-gauge stainless-steel
The hindpaws of mice were held up and the front paws put on a
tested in three nonconsecutive cycles. Stepping was videotaped and
scored by a blind observer afterward. The numbers of left and right paw
steps were counted.
mice received saline or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) (Sigma-Aldrich) at dosages of 4 ? 2 mg/kg or 4 ? 5 mg/kg
intraperitoneally at 2 h intervals (n ? 5). One week later, mice were
perfused with 4% paraformaldehyde and cryoprotected with 30% su-
crose. Brains were sliced on a cryostat. Silver staining was done with FD
NeuroSilver Kit (FD NeuroTechnologies, Ellicott City, MD).
L-DOPA treatment. Four weeks after DOX withdrawal, mice were
divided into two groups to receive daily intraperitoneal injections of
L-DOPA (300 mg/kg, Sigma-Aldrich) plus benserazide (100 mg/kg,
Sigma-Aldrich) or saline for 4 weeks. Benserazide is an inhibitor of pe-
426 • J.Neurosci.,January9,2008 • 28(2):425–433Chenetal.•CytosolicDACausesNeurodegenerationinMice
ripheral DOPA decarboxylase to prevent the conversion of L-DOPA in
of fourth week of L-DOPA treatment, mice were perfused, and unbiased
stereological cell counting was performed to assess neuron number in
Glutathione measurement. Four weeks after DOX withdrawal, mice
on ice and then stored in a ?80°C freezer. Reduced glutathione (GSH)
and oxidized glutathione (GSSG) content were measured using a gluta-
thione assay kit (Cayman Chemical, Ann Arbor, MI) (n ? 5). GSH and
GSSG contents were normalized to protein content and presented as
nmol/mg protein. Protein was quantified with BCA protein quantifica-
tion kit (Fisher).
two-tailed Student’s t tests were used when genotype or DOX treatment
was the only grouping variable. ANOVA were used when additional
factors were considered. Repeated-measures ANOVA were used when
data were collected in multiple trials. In figures, * indicates p ? 0.05, **
indicates p ? 0.01, and *** indicates p ? 0.001. The exact p value (if it is
not ?0.001) is indicated in figure legends.
The transgenic mice with inducible forebrain DAT overexpres-
sion were engineered using the tetracycline inducible system
(Gossen and Bujard, 1992) (Fig. 1A). A tetO-DAT knock-in line
trol of the tetO promoter. In homozygous tetO-DAT mice, DAT
expression is reduced to ?5% of WT expression levels (data not
shown). The tetO-DAT mice were then crossed with the well
al., 1996) (from The Jackson Laboratory, Bar Harbor, ME) to
generate CamDAT mice. In these mice, ectopic DAT expression
is driven by tTA-dependent tetO activity in a forebrain-specific
from the remaining wild-type allele.
To characterize DAT expression, homozygous tetO-DAT
ate CamDAT mice and TetDAT mice. All mice were kept on
dietary DOX (200 mg/kg rodent chow, from Bio-Serv) from ges-
tation until the experiments started, inhibiting ectopic DAT ex-
tubercle, olfactory bulb, cortex, and hip-
pocampus (for lower-power photomicro-
magnification image at the cellular level,
see supplemental Fig. 1, available at www.
jneurosci.org as supplemental material).
Ectopically expressed DAT protein was
erately expressed in the cortex and hip-
pocampus. As expected for a protein en-
riched in axon terminals, DAT was most
abundantly present in two main striatum
efferent nuclei, globus pallidus and sub-
stantia nigra pars reticulata, that receive
inputs from striatal D1-positive and D2-
positive medium spiny neurons, respec-
tively (Fig. 1C). DAT protein was also ex-
pressed on cell bodies of neurons in
striatum of CaMDAT mice (supplemental
Fig. 1, available at www.jneurosci.org as supplemental material).
the forebrain, DAT protein levels in the CamDAT mice after 3
weeks of DOX withdrawal were approximately three times the
over in the CamDAT mice and WT mice, which is supported by
withdraw, striatal tissue dopamine content decreased by 76%,
whereas DOPAC and HVA increased by 68 and 368%, respec-
66%, whereas DOPAC and HVA increased by 38 and 192%, re-
spectively. In contrast, in the control tissue cerebellum, where
changed by DOX withdrawal, whereas DOPAC and HVA levels
increased by 31 and 47%, respectively. To exclude the possibility
rons in midbrain, we quantified dopamine neurons by unbiased
stereological cell counting. The dopamine neuron numbers in
SNc were comparable between the DOX group and no DOX
group of CamDAT mice (5938 ? 535 and 6300 ? 364, respec-
tively; data are given as mean ? SEM).
“sink” that quickly sequesters presynaptic dopamine (Fig. 2A)
and that dopamine is turned over at a high rate, a phenomenon
that has been described in the DAT knock-out mice (Jones et al.,
1998). The increased dopamine metabolites in the cerebellum
suggest diffusions from dopamine terminals. This is consistent
with the observed increases of HVA and DOPAC in the cerebel-
lum of L-DOPA-treated animals (el Gemayel et al., 1986).
Ectopic DAT expression in the forebrain had severe behavioral
consequences (Fig. 3A). If not treated with dietary DOX, Cam-
DAT pups were smaller, and most of them died ?1 week after
shown). Endogenous DAT mRNA was detected in SNc. In control mice (CTL), no DAT mRNA expression was detected in the
Chenetal.•CytosolicDACausesNeurodegenerationinMiceJ.Neurosci.,January9,2008 • 28(2):425–433 • 427
weaning. If CamDAT mice were treated
with DOX from gestation stage on, they
were indistinguishable from TetDAT
mice. However, if DOX treatment was
withdrawn at any time point, most mice
died 4–8 weeks afterward (Fig. 3A).
We noted that lethality in CamDAT
mice after DOX withdrawal was preceded
by severe hypolocomotion and lack of
feeding. CamDAT mice developed se-
verely impaired locomotor activity after 2
weeks of DOX withdrawal (Fig. 3B). We
monitored animals’ body weight and food
intake throughout DOX withdrawal pe-
riod. Significant body weight loss was ob-
(Fig. 3C) or CamDAT mice on DOX (data
not shown). Body weight loss was appar-
ently caused by lack of feeding (Fig. 3D).
However, when food intake was assessed
using easily accessible food (20 mg pellets
consumed more than the control group
(Fig. 3E). Therefore, the lack of feeding is
most likely a result of motor dysfunction
rather than the lack of appetite.
To explore whether neuropathological
changes might underlie the behavioral ab-
normalities of CamDAT mice, mice were
perfused, and brain sections were immuno-
stained with a neuron-specific marker
(NeuN) after 4 weeks of DOX withdrawal.
We first measured the volume of striatum
and cortex as well as olfactory bulb. We
found significant striatal and cortical atro-
phy in CamDAT mice after DOX with-
drawal compared with those kept on DOX
(Fig. 4A,C). These areas have both ectopic
but has no significant dopamine afferents
merular layer do not project to the granule
cell layer, where ectopic DAT expressing
We analyzed the striatal neuropathol-
ogy further by unbiased stereology counting of NeuN-positive
cells. We found 10% neuron loss in the striatum of CamDAT
mice after DOX withdrawal compared with those kept on DOX
(Fig. 4D). Although the neuronal loss strongly suggested dopa-
We investigated ultrastructural evidence for neurodegenera-
tion by transmission electron microscopy. Striatal neurons
showed various degrees of abnormal morphologies. Some af-
fected neurons only displayed dilated organelles, such as Golgi
apparatus and mitochondria. Other affected neurons displayed
prominent cytoplasmic organelle disintegration, although the
morphology of their nucleus remained relatively normal. The
more severely affected neurons displayed vacuolization and dis-
integration of the nucleus (Fig. 4F). The latter two types of
pathology account for ?50% of striatal neurons. The typical
apoptotic morphology, such as cell shrinkage, chromatin con-
densation, membrane blebbing, and phagocytosis, was lacking.
We also used apoptotic markers to examine potential apoptosis.
Likewise, we found no increase of active caspase-3 or terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling-positive neurons in CamDAT mice with ectopic
DAT expression (data not shown).
H2O2, and other reactive species in postsynaptic neurons. B, After 3 weeks of DOX withdrawal, striatum and cortex dopamine
428 • J.Neurosci.,January9,2008 • 28(2):425–433Chenetal.•CytosolicDACausesNeurodegenerationinMice
To explore further potential functional changes in striatal
sion, which are neuropeptides specifically found in D1-positive
striatum did not reveal differences in expression levels 4 weeks
after DOX removal in CamDAT mice compared with control
group (supplemental Fig. 2, available at www.jneurosci.org as
types in D1-positive and D2-positive neurons were preserved,
although there were profound changes in morphology.
In addition to neurodegeneration, immunohistochemical
staining with an antibody against GFAP revealed obvious astro-
gliosis in striatum and cortex of CamDAT mice after 4 weeks of
DOX withdrawal but not in control mice (Fig. 4B). Although
neurons by immunostaining and Western blot (supplemental
Fig. 3, available at www.jneurosci.org as supplemental material),
suggesting that neurodegeneration in CamDAT mice is likely to
be ?-synuclein independent.
To further investigate the mechanism of neurodegeneration, we
idation to dopamine-quinone, which reacts with cysteine resi-
dues on proteins to form cysteinyl-dopamine and cysteinyl-
DOPAC conjugates (Hastings and Zigmond, 1994; Sulzer and
Zecca, 2000; Barzilai et al., 2003). To assess the abundance of
quinone-modified proteins, HPLC analyses of 5-cysteinyl-
dopamine and 5-cysteinyl-DOPAC levels were used to assess for
quinone modified protein (Hastings et al., 1996; LaVoie and
Hastings, 1999). We found significant increases of dopamine-
quinone by 527% and DOPAC-quinone by 149% evidenced by
modified cysteine residues in CamDAT mice after 3 weeks of
DOX withdrawal compared with animals kept on DOX. DOPA-
quinone modified cysteine residues were unchanged (Fig. 5A).
Similarly, in cerebral cortex, we found increases of dopamine-
quinone by 138% and DOPAC-quinone by 88% noted by mod-
ified cysteine residues. DOPA-quinone modified cysteine resi-
change was found for dopamine, DOPA, or DOPAC-quinone
modified cysteine residues (Fig. 5A).
In addition to reactive quinones, dopamine metabolism also
produces H2O2and superoxide, which may contribute to oxida-
tive stress. Therefore, glutathione content was examined. Com-
pared with control mice, total glutathione (GSH?GSSG), GSH,
( p ? 0.01), and 27% ( p ? 0.05), respectively, in CamDAT mice
cient to cause mitochondria damage in brain (Jain et al., 1991). In
cerebral cortex and cerebellum, total glutathione (GSH?GSSG),
ropathology, L-DOPA would be predicted to accelerate such a
process by boosting dopamine supply. Four weeks after DOX
withdrawal, we treated CamDAT mice with another 4 weeks of
daily intraperitoneal injections of 300 mg/kg L-DOPA plus 100
mg/kg benserazide, and body weight was monitored. L-DOPA-
(41.5 vs 25% by the end of fourth week, respectively, n ? 5, p ?
revealed significant NeuN?cell loss in L-DOPA-treated Cam-
DAT mice compared with saline-treated CamDAT mice (n ? 4,
12% loss, p ? 0.05) (Fig. 6B).
Striatal neurons in CamDAT mice express ectopic DAT without
the capacity to synthesize dopamine, and therefore the accumu-
lation of cytoplasmic dopamine in these mice depends on supply
of extracellular dopamine. To assess whether the behavioral im-
toxicity effect), we performed unilateral lesions of the medial
forebrain bundle by 6-hydroxydopamine before DOX with-
drawal to remove the major source of dopamine supply for stri-
ual forelimbs was assessed weekly by stepping behavior on a mo-
torized treadmill. Stepping of the contralateral forelimb con-
trolled by the lesion side was significantly impaired (16% of that
deficit resulting from dopaminergic deafferentation (Chang et
al., 1999). The 6-OHDA-lesion paradigm used in current study
damages ?90% dopamine neurons, which is confirmed by im-
munostaining with tyrosine hydroxylase antibody (data not
sive body weight loss in CamDAT mice after DOX withdrawal (n ? 6, repeated-measures
DAT mice in their home cages consumed very little regular rodent chow (n ? 5 per group,
individually housed, unpaired t test, p ? 0.001). E, Two weeks after DOX withdrawal, when
Chenetal.•CytosolicDACausesNeurodegenerationinMiceJ.Neurosci.,January9,2008 • 28(2):425–433 • 429
shown). After DOX withdrawal, stepping
intact side declined drastically (Fig. 7)
(stepping of the ipsilateral forelimb con-
drawal was used as baseline). In contrast,
there was no further deterioration of the
gradual partial improvement of stepping
of the contralateral forelimb controlled by
the lesion side was noted (Fig. 7). These
data suggest that the stepping dysfunction
of the intact side after DOX withdrawal
was caused by uptake of released dopa-
mine from presynaptic terminals, and
such toxicity was spared in the lesion side,
where the source of dopamine was re-
moved. The partial improvement of the
contralateral limb is intriguing. There are
havioral adaptation after acute damage.
The second possibility is that forced use of
this particular limb after drastic deteriora-
covery from the lesion. Functional and ana-
tomical recovery after 6-OHDA lesion
been reported by other groups (Tillerson et
gether, the above data strongly suggest that
the behavioral abnormality in CamDAT
MPTP-treated mice usually develop dopamine neuron degener-
ation and are commonly used as PD models (Przedborski et al.,
2004). The selective loss of dopamine neurons in the MPTP
model depends on DAT expression in dopamine neurons (Gai-
CamDAT mice are especially vulnerable to dopamine-induced
neurodegeneration. To examine whether they are also especially
vulnerable to MPTP and to confirm that their ectopically ex-
neurodegeneration in striatum, cortex, and hippocampus was ex-
amined. Two weeks after DOX withdrawal, a single dose of MPTP
CamDAT mice kept on DOX remained active. Therefore, we low-
1 week after MPTP treatment. Using silver staining, degenerative
found in saline-treated mice or in low-dose MPTP-treated Cam-
ectopically expressed DAT in striatal neurons are functional, and
these neurons are more susceptible to MPTP treatment than dopa-
mine neurons in substantia nigra, which have protective mecha-
able to isolate dopamine as the determining factor in causing
neurodegeneration. In our model, dopamine supply determines
the progression of neurodegeneration, which is accelerated by
L-DOPA treatment and attenuated by removing dopaminergic
afferents. Therefore, we provide definitive evidence that chronic
produce neurodegeneration in vivo. Moreover, severe neuropa-
In the transgenic mice, striatal neurons were engineered to
take up extracellular dopamine released from dopaminergic ter-
minals without acquiring intrinsic regulatory mechanisms typi-
cally found in dopamine neurons. Therefore, we were able to
isolate the contribution of dopamine without introducing other
confounding factors. In normal dopamine neurons, there are
major regulatory mechanisms that include the VMAT2 and reg-
ulated dopamine synthesis and activity. Those mechanisms
pamine within relatively nontoxic levels (Zigmond et al., 1989;
Kumer and Vrana, 1996; Eisenhofer et al., 2004). Therefore, it
may require decades of exposure to low levels of cytosolic dopa-
pathology to take place.
that the detrimental effects of dopamine in the present animal
model are mediated via dopamine metabolism and oxidation.
H2O2and other reactive oxidative species produced naturally in
dopamine degradation pathways may oxidize proteins, lipids,
and DNA, which ultimately cause cell dysfunction and neurode-
striatum. n ? 6 per group. D, NeuN-positive cells in the striatum decreased by 10% (n ? 4, unpaired t test, p ? 0.04). E,
430 • J.Neurosci.,January9,2008 • 28(2):425–433Chenetal.•CytosolicDACausesNeurodegenerationinMice
generation (Stokes et al., 1999). Of particular importance is the
modification of cysteinyl residues by dopamine- and DOPAC-
quinones. Cysteinyl residues, the prime target of electrophilic
quinones, often reside at the active sites of proteins, and thus,
their covalent modifications by quinones could inactivate func-
tion, alter conformation, and promote aggregation of proteins
(Graham, 1978). One of the main defense mechanisms for such
detrimental effects is glutathione. Glutathione is ready to bind
dopamine- and DOPAC-quinones by their thiol group so that
other proteins are spared of their detrimental modifications. In
agreement with cytosolic dopamine toxicity in our transgenic
5-cysteinyl-DOPAC and decrease of total glutathione, similar to
what have been found in PD patients (Schulz et al., 2000).
Dopamine toxicity-induced neurodegeneration may involve
?-synuclein, including its oxidative modification. It has been
found that dopamine and other catechols inhibit ?-synuclein
fibrillization and lead to accumulation of ?-synuclein protofi-
brils. These include studies in both cell culture (Montine et al.,
dopamine itself are responsible for modifying ?-synuclein,
mainly by noncovalent modification on125YEMPS129motif by
dopaminochrome, DA-quinone, DOPA-quinone, and DOPAC-
quinone. Therefore, we investigated the possibility that
that neurodegeneration in CamDAT mice is likely to be
Our data show severe motor impairment after DOX removal
in CamDAT mice. Because striatal neuron loss is modest, striatal
neuron dysfunction is more likely to be the cause of their motor
impairment. Our EM ultrastructural data indicate that ?50%
Decreasedglutathionecontentinbraintissues.Totalglutathione(GSH?GSSG),GSH,andGSSGwere26%( p?0.01),29%( p?0.01),and27%( p?0.05)lower,respectively,instriatumof
DOX withdrawal, CamDAT mice were injected daily with 300 mg/kg L-DOPA plus 100 mg/kg
L-DOPA accelerates neurodegeneration and body weight loss. Four weeks after
Motor dysfunction in CamDAT mice is dopamine supply dependent. DOX treat-
Chenetal.•CytosolicDACausesNeurodegenerationinMice J.Neurosci.,January9,2008 • 28(2):425–433 • 431
striatal neurons have degenerative mor-
phologies. Low striatal dopamine content
might be another contributor. However,
our unilateral lesion data clearly indicate
nificant contributor than dopamine defi-
ciency to the motor impairment pheno-
type in CamDAT mice.
The major pathological phenotype of
our mice bears some similarities to that of
models of Huntington’s disease, which is
characterized by neuropathology in stria-
tal medium spiny neurons. Interestingly,
extracellular dopamine has been shown to
worsen neuronal loss caused by mutant
huntingtin in both cultured cells and mice
models (Hastings et al., 1996; Jakel and
Maragos, 2000; Cyr et al., 2003, 2006;
Charvin et al., 2005; Tang et al., 2007). In
comparison, the CamDAT mouse is a cy-
tosolic dopamine toxicity model. There-
fore dopamine is likely to be a toxic agent,
whether elevated inside or outside of
Our data highlight the importance of cellular regulatory
mechanisms for cytosolic dopamine homeostasis in preventing
dopamine induced neurotoxicity and the importance of protec-
tive biochemical pathways such as glutathione. Under normal
conditions, those protective mechanisms along with others such
as the ubiquitin-proteasome system might work in concert to
prevent dopamine neurons from degeneration. However, the
ple, the disruption of dopamine vesicular storage by ?-synuclein
has been suggested (Conway et al., 2001; Lotharius and Brundin,
2002; Cooper et al., 2006). DJ-1-deficient mice have been shown
to exhibit elevated DAT function and total tissue dopamine con-
to have increased extracellular dopamine (Bonifati et al., 2003;
one of the targets modified by dopamine quinone (LaVoie et al.,
2005). Synuclein protofibrils can also be modified and stabilized
by catechols related to dopamine but not other compounds
(Conway et al., 2001).
Our results have significant clinical implications for the po-
Results from the recent large-scale clinical trials are mixed.
L-DOPA improved the signs and symptoms of the disease, sug-
gesting neuroprotection. However, the same trial showed re-
duced uptake of DAT substrates, suggesting more rapid dopa-
mine terminal loss in L-DOPA-treated patients (Rascol et al.,
2000; Parkinson Study Group, 2002; Whone et al., 2003; Hollo-
way et al., 2004; Fahn, 2005). Our L-DOPA treatment data indi-
dopamine replacement therapies will accelerate neurodegenera-
tion. This point is also relevant for many of the PD gene therapy
approaches that involve the expression of tyrosine hydroxylase
(Kang et al., 2001; Liste et al., 2004). Virus-infected neurons
(mostly striatal neurons) and most engineered cells (e.g., fibro-
blasts) do not have the complete cellular machinery to protect
them from cytosolic dopamine toxicity. Therefore, reducing cy-
tosolic dopamine toxicity might be necessary to prevent any po-
tential damages associated with such therapies.
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