N-Acetylcysteine Prevents Loss of
Dopaminergic Neurons in the
Ari E. Berman, Ph.D., Wai Yee Chan, Ph.D., Angela M. Brennan, Ph.D.,
Reno C. Reyes, Ph.D., Brittany L. Adler, B.S., Sang Won Suh, Ph.D.,
Tiina M. Kauppinen, Ph.D., Ylva Edling, Ph.D., and Raymond A. Swanson, M.D.
Objective: Dopaminergic neuronal death in Parkinson’s disease (PD) is accompanied by oxidative stress and preceded
by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of
animal models that mimic these features and demonstrate an age-related progression. The EAAC1?/?mouse may be
useful in this regard, because EAAC1?/?mouse neurons have impaired neuronal cysteine uptake, resulting in reduced
neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes
in nigral dopaminergic neurons in the EAAC1?/?mouse, and (2) use the EAAC1?/?mouse to evaluate N-
acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD.
Methods: Wild-type mice, EAAC1?/?mice, and EAAC1?/?mice chronically treated with N-acetylcysteine were
evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities.
Results: EAAC1?/?mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars
compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by
substantially reduced in mice that received N-acetylcysteine.
Interpretation: These findings suggest that the EAAC1?/?mouse may be a useful model of the chronic neuronal
oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus
for clinical evaluation of glutathione repletion in PD.
ANN NEUROL 2011;69:509–520
neurons of the substantia nigra pars compacta (SNc).
Between 5% and 10% of PD can now be attributed to
heritable genetic mutations, but the cause of the more
common, sporadic PD remains elusive. Several lines of
evidence suggest that oxidative stress and depletion of
glutathione, a major endogenous antioxidant, contributes
to neuronal death in both hereditary and sporadic PD.
Oxidative stress and dopaminergic neuronal death are
produced by chemical agents epidemiologically associated
with PD, such as paraquat and rotenone.1,2Postmortem
studies of PD patients show increased lipid and protein
arkinson’s disease (PD) leads to cell death in several
neuronal populations, particularly the dopaminergic
oxidation products in the SNc and markedly reduced lev-
els of glutathione.3,4Moreover, the glutathione depletion
precedes dopaminergic neuronal death in PD5and does
not occur in other neurodegenerative disorders affecting
the SNc,6thus suggesting a specific and causal role for
glutathione depletion in PD pathogenesis.
Development of interventions to slow or prevent
neuronal death in PD has been hindered by a paucity of
animal models of the chronic, sustained neuronal oxidative
stress observed in human PD.7,86-Hydroxydopamine
(MPTP), and other toxins used to generate animal models
of PD produce massive, acute oxidative stress in the
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22162
Received Jul 26, 2009, and in revised form Jul 3, 2010. Accepted for publication Jul 13, 2010.
Address correspondence to Dr Swanson, Neurology VAMC, 4150 Clement St., San Francisco, CA 94121. E-mail: email@example.com
Ari E. Berman and Wai Yee Chan contributed equally to this work.
Current address for Dr Chan: Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb Company, Princeton, NJ 08534.
From the Department of Neurology, University of California, San Francisco and the San Francisco Veterans Affairs Medical Center, San Francisco, CA.
Additional Supporting Information can be found in the online version of this article.
C 2011 American Neurological Association 509
targeted neuronal populations, leading to rapid cell death
and motor abnormalities. These models generate a pheno-
type resembling human PD, but they do not recapitulate
the chronic oxidative stress and slow neurodegeneration
that occurs over decades in human PD. The EAAC1?/?
mouse may be a more useful model in this respect because
this mouse strain exhibits chronic, neuron-specific oxidative
stress.9EAAC1 (also termed EAAT3 and SLC1A1) is an
excitatory amino acid transporter expressed selectively by
neurons in the central nervous system (CNS).10,11It is the
primary route for neuronal uptake of cysteine,9,12,13the
rate-limiting substrate in glutathione synthesis.14Mice lack-
ing EAAC1 have decreased neuronal glutathione content,
increased markers of neuronal oxidative stress, and a slow,
age-dependent reduction in overall brain size9; however,
the effect of EAAC1 deficiency on SNc dopaminergic neu-
rons has not previously been reported.
The striking depletion of glutathione in dopaminer-
gic neurons in PD, coupled with the importance of glu-
tathione for neuronal survival, has led several authors to
suggest that preventing this glutathione depletion may be
neuroprotective in PD.15,16Glutathione is involved in
the elimination of peroxides and nitrosylated proteins in
both the cytosol and mitochondria. N-acetylcysteine
(NAC) is a cell membrane–permeable form of cysteine
that can cross the blood-brain barrier and replete neuro-
nal glutathione content.9,17,18NAC is well-tolerated and
currently in clinical use for other indications.19Here,
using the EAAC1?/?mouse as a model of chronic neuro-
nal oxidative stress, we show an age-dependent loss of
SNc dopaminergic neurons in the EAAC1?/?mouse,
and further show that this neuronal loss is reduced by
oral administration of NAC. These results provide a ra-
tionale for evaluating glutathione repletion as a disease-
modifying therapy for PD.
Subjects and Methods
Animal and human studies were performed in accordance with
protocols approved by institutional review committees at the
San Francisco Veterans Affairs Medical Center. Reagents were
obtained from Sigma-Aldrich except where noted.
The EAAC1?/?mice have exon 1 of the EAAC1 gene dis-
rupted by a neomycin resistance (NEO) cassette.20EAAC1?/?
mice were obtained from Miltenyi Biotec GmbH (Bergisch
Gladbach, Germany) and subsequently outbred to wild-type
(WT) CD-1 mice for more than 10 generations, as described
previously.9WT mice were maintained using the offspring from
the latter outcrosses. Breeding stock from the WT and
EAAC1?/?mice were subsequently intercrossed at least once ev-
ery 8 generations to prevent genetic drift, in accordance with
the Banbury Conference recommendations.21Genotypes were
confirmed by polymerase chain reaction (PCR) of tail DNA,
using 3 primers that together amplify sequences unique to the
WT and allele and to the inserted NEO cassette of the dis-
GATGTGACT-30) was common to both sequences. The WT
reverse primer (50-CAGGGTGGAGAGCA-GCAG-30) produces
a 63-bp PCR product, and the NEO reverse primer (50-
PCR product (Supporting Information Fig S1). Genotyping
gels were run on 3% agarose gels using a 100-bp ladder (Prom-
ega) for size determinations.
Administration of N-Acetylcysteine
One cohort of mice was fed NAC in the drinking water begin-
ning 1 week after weaning. The water contained 4mg/ml NAC
for the first 6 months, then 2mg/ml for the remainder of the
study. The NAC solution was pH-adjusted to 6.0 to match the
pH of the water normally given to the animals in the animal
facility, and the solution was replaced twice weekly to limit
autoxidation. For studies of NAC’s effect on neuronal glutathi-
one content, 4-month-old EAAC1?/?mice were given 2mg/ml
NAC in drinking water for 7 days.
Immunohistochemistry and Confocal Imaging
Mice were perfusion-fixed with 4% formaldehyde. The brains
were postfixed in 4% formaldehyde at 4?C, followed by cryopro-
tection in 20% sucrose and freezing. The frozen brains were
coronally sectioned and immunostained using the following anti-
bodies: rabbit anti-EAAC1 (1:200, gift from Dr. J Rothstein,
Johns Hopkins University); sheep anti-tyrosine hydroxylase
(1:400, Chemicon, AB1542); mouse anti-NeuN (1:250, Chemi-
con, MAB377); rabbit anti-nitrotyrosine (anti-nTyr; Chemicon,
1:100, AB5411); and rabbit anti–ionized calcium binding
adaptor molecule 1 (Iba1; 1:400, Wako, 019-19741). Antibody
binding was detected using fluorescent donkey anti-rabbit, anti-
mouse, and anti-sheep immunoglobulin G (IgG) (all 1:250; Invi-
trogen) as previously described.9,22
captured using a Zeiss LSM 510 Meta confocal microscope using
sequential scans for double-stained and triple-stained samples.
Control sections prepared with no primary or no secondary anti-
bodies showed no detectable signal. Controls also established
that signal attributed to each of the fluorophores was completely
absent when omitted from the staining procedure.
Confocal images were
Neuronal Cell Counts
Quantification of SNc dopaminergic neurons was performed
using a stereological approach.23Sequential 30-lm cryostat sec-
tions were obtained spanning the rostral-caudal extent of the
SNc. Cell counts were performed on 4 evenly-spaced sections
from each brain, with the most caudal section analyzed at
Bregma ?3.60 mm, and the most rostral section at Bregma
?3.00 mm.24The sections were stained for tyrosine hydroxy-
lase (TH) and photographed with a confocal microscope using
an optical width of 12.4 lm and a z-step of 6.1 lm. The SNc
was identified by TH staining and standard landmarks.24Cell
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510Volume 69, No. 3
counting was performed on an optical z-section 18–19 lm
from the upper surface of each physical section to prevent bias
due to cell loss from the cut surfaces. Cells were counted
throughout the entire SNc on the optical sections, rather than
on random samples of the sections, to avoid estimation errors
introduced by sampling. TH-positive neurons were counted
only if the nucleus was fully contained in the optical section, as
evidenced by a circumscribed lacune in TH staining of the cell
soma. This served to exclude any bias in cell counts that could
otherwise result from changes in neuronal soma size. Counts
from each section were summed for each animal and expressed
as the mean number of cells per bilateral midbrain section. Cell
counts were performed twice, by 2 individuals blinded to the
mouse treatment group, and the independently obtained cell
counts were averaged to produce a single value for each animal.
The cell counts obtained by the different observers were within
9.1 6 7.4% (mean 6 SD) of one another.
Quantification of nTyr and Reactive Thiols
Evaluations were performed on a coronal section from each
mouse corresponding to Bregma ?3.40 mm.24All sections were
immunostained together to minimize differences introduced by
the staining procedure. nTyr staining of dopaminergic neurons
was measured by first using the TH staining to define regions of
interest on confocal photomicrographs of each section, and then
measuring nTyr staining intensity selectively in these regions of
interest. The intensity of nTyr staining over all TH-positive SNc
neurons was averaged to generate a single value for each brain.
Reactive thiols in TH-positive neurons were analyzed by
a similar method.9Fixed sections were first incubated for 24
hours at 4?C in a serum-free solution of 2.5 lM Alexa Fluor
488 C5-maleimide (Invitrogen). C5-maleimide binds to reactive
thiols, of which glutathione is the dominant species. The sec-
tions were subsequently immunostained for TH to identify do-
paminergic neurons. The TH staining was used to define
regions of interest on confocal photomicrographs of each sec-
tion, and then the intensity of C5-maleimide staining was
measured within these regions of interest. The intensity of C5-
maleimide staining over all TH-positive SNc neurons was aver-
aged on 4 evenly spaced sections spanning the SNc to generate
a single value for each brain.
Measurements of Biogenic Amines
Analyses were performed on striata isolated from WT and
EAAC1?/?mice age 9–11 months. The striata were isolated on
a chilled glass plate and homogenized in 0.1 M perchloric acid.
Dopamine and related metabolites were measured by the
CMN/KC Neurochemistry Core Lab at Vanderbilt University,
using high-performance liquid chromatography with internal
standards and electrochemical detection. Results were normal-
ized to protein content.
Evaluations were performed on the coronal section from each
mouse corresponding to Bregma ?3.20mm, which is near the
rostral-caudal midpoint of the SNc.24Sections from each brain
were immunostained simultaneously for TH and Iba1. To opti-
mize imaging of microglial processes, a z-stack of 20 overlapping
2-lm-thick confocal images was acquired through each section
and then compressed to generate a single image. Microglia mor-
phology was evaluated as described previously,22,25using the crite-
ria described and illustrated in Supporting Information Figure S2.
Western blots were performed as described previously.9,22In brief,
whole-brain lysates were homogenized in ice-cold radioimmuno-
precipitation assay buffer and centrifuged at 5,000 rpm. The su-
pernatant was added to sample buffer containing b-mercaptoeth-
anol (final concentration 7.5%), electrophoresed on 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis gels, trans-
ferred to polyvinylidene fluoride membranes, and incubated with
antibody to nitrated a-synuclein (nSyn14; Chemicon, 1:5,000).
After membrane stripping, the membranes were reprobed with
antibody to EAAC1 (Alpha Diagnostics, 1:1,000) to confirm the
genotype. Comparable loading was confirmed by staining for b-
actin (Sigma, 1:6,000). Antibody binding was visualized with the
chemiluminescence detection (ECL Plus, GE Healthcare).
Studies of motor function were conducted in a quiet, dimly-lit
room. The open field test of spontaneous locomotor activity26
was performed by placing mice in a Plexiglas enclosure equipped
with 2 rows of infrared photocell panels interfaced with a com-
puter. The lower row detected horizontal motion, and the higher
row detected vertical motion (rearing). On each of 3 consecutive
days, open field activity was recorded for 10 minutes after an ini-
tial 1-minute adaptation period. Rotarod testing26,27was per-
formed by placing mice onto a rotating dowel, with the speed of
rotation increasing at a constant rate over the course of the trial.
The time each animal was able to stay on the dowel was meas-
ured on 3 trials per day for 3 consecutive days. Mice were
allowed a minimum of 1 minute recovery between each trial. For
the pole test of agility26,28mice were placed facing upward near
the top of a cloth-covered vertical pole, 30cm high and 1.5cm in
diameter. The time required for the animals to turn around and
climb to the bottom was recorded. Tests were performed twice in
succession on 3 consecutive days, and the mean time recorded
after discard of the furthest outlying time point. Trials in which
the animal was unable to successfully turn around and reach the
bottom were terminated at 3 minutes.
Formalin-fixed, paraffin-embedded human midbrain samples
from 4 PD brains and 4 control brains were acquired from the
Harvard Brain Tissue Resource Center. All samples were from
individuals ranging in age from 72 to 79 years old, and were
obtained after postmortem intervals of 15–28 hours. The PD
brains all showed Lewy bodies and moderate to severe cell loss
in the SNc. Sections (5lm) through the SNc and the red nu-
cleus were prepared on a sliding microtome. Paraffin was
removed by xylene immersion, and rehydration was performed
in a graded ethanol wash series. The samples were then boiled
Berman et al: NAC Prevents SNc Neuronal Loss in EAAC1–/–Mice
for 10 minutes in citrate buffer to achieve heat-induced epitope
retrieval.29Immunostaining for TH and EAAC1 were per-
formed as described for the mouse tissues.
Microglial activation data were analyzed with a Student t test.
Cell counts, biogenic amine, C5-maleimide staining, nTyr, and
behavioral data were analyzed by one-way analysis of variance
(ANOVA) followed by the Bonferroni post-hoc test for compar-
isons between multiple groups. Where cell count and nTyr data
are displayed in more than 1 figure, statistical analyses
accounted for all multiple comparisons.
Progressive Loss of SNc Dopaminergic
Neurons in EAAC1?/?Mice
The number of dopaminergic neurons in the SNc was
evaluated in WT and EAAC1?/?mice at 3, 9, and 12
months of age (Fig 1). No differences were found at 3
months, but a significant, 41 6 10% decrease in the
EAAC1?/?brains was apparent by age 12 months (see
Fig 1). Microglia assume an activated morphology in the
presence of injured and dying neurons, and activated
microglia are present in the SNc in PD.30To comple-
ment the studies of neuronal loss, brain sections were
stained for the microglia marker, Iba1, to assess micro-
glial morphology in 12-month old EAAC1?/?and WT
mice. The EAAC1?/?mice showed increased Iba1 im-
munoreactivity and activated morphology in the SNc
and surrounding area (Fig 2).
Despite these changes in the SNc, measurements of
dopamine and related biogenic amines in the striatum
showed only nonsignificant reductions in the EAAC1?/?
mice (Supporting Information Fig S3). Immunostaining
for tyrosine hydroxylase in the striatum similarly showed
FIGURE 1: Progressive loss of SNc dopaminergic neurons in EAAC12/2mice. Confocal images of the dorsal midbrain of WT
and EAAC12/2mice, with dopaminergic neurons stained green (anti-TH). Insets show magnified view of boxed areas. Graphs
show quantification of dopaminergic neurons in the SNc at 3, 9, and 12 months of age. Bar 5 200 lm. **p < 0.01; n 5 3–7.
SNc 5 substantia nigra pars compacta; SNr 5 substantia nigra pars reticulata; VTA 5 ventral tegmental area; WT 5 wild-type.
ANNALS of Neurology
512 Volume 69, No. 3
no significant reduction (not shown), presumably because
of the compensatory sprouting from surviving SNc dopa-
minergic neurons that occurs with chronic lesions.31,32
Increased Oxidative Stress in EAAC1?/?
SNc Dopaminergic Neurons
Glutathione is important for metabolism of peroxides, ni-
tric oxide, and other oxidant species. EAAC1?/?mice
have reduced neuronal glutathione content due to the lack
of EAAC1-mediated neuronal cysteine uptake.9,13,33,34To
assess the level of oxidative stress in EAAC1?/?midbrain
dopaminergic neurons, brain sections were immuno-
stained for the presence of nTyr-modified proteins.35SNc
dopaminergic neurons in the EAAC1?/?mice showed an
increase in nTyr immunoreactivity (Fig 3A, B). As a sec-
ond measure of oxidative stress, nitrosylated a-synuclein
was measured in WT and EAAC1?/?brains. Aggregated
a-synuclein fibrils are the primary component of Lewy
bodies found in PD, and the formation of these aggregates
is accelerated by nitrosylation.36Nitration and oxidation
of a-synuclein produce cross-linked a-synuclein oligomers
that are stable to sodium dodecyl sulfate.37Western blots
of brain tissue from WTand EAAC1?/?mice confirmed a
striking increase in nitrosylated a-synuclein, primarily as
dimers and higher-order aggregates, in the aged EAAC1?/?
mice (Fig 3C).
EAAC1 Expression in Mouse and Human
SNc Dopaminergic Neurons
The distribution of EAAC1 expression in the mouse
midbrain was evaluated by immunostaining, with neuro-
nal nuclei identified by NeuN expression and dopami-
nergic neurons identified by tyrosine hydroxylase (TH)
expression. EAAC1 was diffusely distributed in the mid-
brain, consistent with its ubiquitous expression over neu-
ronal cell bodies and processes.10,11However, there was
higher EAAC1 expression over dopaminergic (TH-posi-
tive) neurons (Fig 4A), in agreement with previous
reports.11,33Human postmortem midbrain sections were
similarly stained for EAAC1 and TH, but for technical
reasons the anti-NeuN antibody could not be used with
the postmortem tissue. As in the mouse brain, normal
human brain showed increased EAAC1 expression in do-
paminergic (TH-positive) neurons (see Fig 4B). The
EAAC1 signal in the surviving dopaminergic neurons of
the PD brain sections was at least as great as in the nor-
mal brain sections, but variability in the immunostaining
quality in the postmortem tissue precluded a quantifiable
Oral NAC Restores Reactive Thiol Content in
EAAC1?/?SNc Dopaminergic Neurons
NAC is a membrane-permeable form of cysteine that has
previously been shown to facilitate neuronal glutathione
synthesis in neuron cultures and in mouse hippocampal
neurons in situ.9,18To confirm that oral NAC could
replete glutathione indopaminergic
EAAC1?/?mice were fed NAC in drinking water for 7
days. The content of reactive thiols, of which glutathione
is the major species, was evaluated by histochemical
staining with C5-maleimide on SNc brain sections
FIGURE 2: Microglial activation in the SNc of EAAC12/2mice. (A) Confocal images of the dorsal midbrain of WT and EAAC12/2
mice, with microglia stained green (anti-Iba1). Dopaminergic neurons were stained blue (anti-TH) to delineate the SNc. Right-hand
figures show magnified view of microglia in the inset boxed areas. Bar 5 100 lm in low-power view, 50lm in high-power view. (B)
Quantification of microglial activation in the SNc. *p < 0.01; n 5 3. SNc 5 substantia nigra pars compacta; SNr 5 substantia nigra
pars reticulata; VTA 5 ventral tegmental area; WT 5 wild-type.
Berman et al: NAC Prevents SNc Neuronal Loss in EAAC1–/–Mice
prepared from these mice. Immunostaining for tyrosine
hydroxylase identified dopaminergic neurons and processes
on the same sections. Dopaminergic neurons from the
EAAC1?/?mice showed significantly less C5-maleimide
staining than neurons from WT mice, and this reduction
was reversed in EAAC1?/?mice fed NAC (Fig 5).
NAC Reduces SNc Dopaminergic Cell Loss
A cohort of EAAC1?/?mice was therefore given NAC in
the drinking water from weaning through 12 months of
age to determine whether long-term administration could
prevent neuronal death resulting from chronic oxidative
stress. Brains evaluated at 12 months of age showed less do-
paminergic neuronal loss in the SNc of EAAC1 mice
treated with NAC (Fig 6A). These neurons also showed
less oxidative stress, as indicated by nTyr immunoreactivity,
than neurons in untreated EAAC1?/?mice (see Fig 6B).
NAC Improves Pole Test Performance
Mice with chronic bilateral loss of dopaminergic neurons
generally display little motor dysfunction unless the loss is
very extensive.26Here we compared WT mice, untreated
EAAC1?/?mice, and NAC-treated EAAC1?/?mice
using a battery of tests of designed to detect abnormalities
in the mouse nigrostriatal system: the open field test for
spontaneous activity, the rotarod test of limb dexterity,
and the pole test of balance and coordination.26–28The
open field test and rotarod tests showed no differences
between the treatment groups (data not shown). On the
pole test, however, the untreated EAAC1?/?mice per-
formed significantly worse than the WT mice at age 12
FIGURE 3: Increased oxidative stress in EAAC12/2SNc dopaminergic neurons. (A) Confocal images of SNc from WT and
EAAC12/2mice. Sections are stained for nitrotyrosine (nTyr, green) as a marker of oxidative stress, and for tyrosine
hydroxylase (TH, blue) to identify dopaminergic neurons. Bar 5 50 lm. (B) Quantified data show that the nTyr signal localized to
dopaminergic neurons is increased in EAAC12/2mice. **p < 0.01; n 5 7. (C) Western blots prepared from WT and EAAC12/2
mouse brain show accumulation of nitrated a-synuclein in 12-month-old EAAC12/2brains but not in 12-month-old WT brains. The
2 left-hand lanes were prepared on a different gel than the other lanes. *Denotes a nonspecific band recognized by the antibody
to EAAC1. WT 5 wild-type.
ANNALS of Neurology
514Volume 69, No. 3
FIGURE 4: EAAC1 expression in mouse and human SNc dopaminergic neurons. (A) Sections through mouse SNc
immunostained for NeuN (red) to identify neuronal nuclei; for EAAC1 (green); and for tyrosine hydroxylase (TH, blue) to
identify dopaminergic neurons. Confocal images show dopaminergic neurons to be densely stained for EAAC1, and
nondopaminergic neurons (some denoted by arrows) to be less densely stained. Bar 5 50lm; representative of n 5 4. (B)
Sections through human normal and PD brains immunostained for EAAC1 (green), and for TH (blue) to identify dopaminergic
neurons. The TH-positive dopaminergic neurons show coexpression of EAAC1 in both PD and control brains. Bar 5 50lm;
representative of n 5 4 control and 4 PD brains.
Berman et al: NAC Prevents SNc Neuronal Loss in EAAC1–/–Mice
March 2011 515
months, and the EAAC1?/?mice treated with NAC per-
formed significantly better than the untreated EAAC1?/?
mice (Fig 7).
Neurons do not take up extracellular glutathione directly,
but instead rely primarily on glial-derived cysteine as a
FIGURE 5: Oral N-acetylcysteine restores reactive thiol content of SNc dopaminergic neurons in EAAC12/2mice. (A)
Representative confocal sections through mouse SNc labeled with C5-maleimide (green) to label reactive thiols and
immunostained for tyrosine hydroxylase (red) to identify dopaminergic neurons. Bar 5 50lm. (B) Quantification of C5-
maeimide fluorescence in TH-positive neurons indicates that reactive thiol content of dopaminergic neurons is lower in
EAAC12/2mice than in WT mice, but normalized in EAAC12/2mice treated with oral NAC. *p < 0.05; n 5 4.
ANNALS of Neurology
516Volume 69, No. 3
precursor for glutathione synthesis.14The EAAC1?/?
mouse has impaired neuronal cysteine uptake, resulting
in chronic neuronal oxidative stress and age-dependent
brain atrophy.9Results of the present studies show that
dopaminergic neurons of the SNc are particularly
affected in the EAAC1?/?mouse, with more than 40%
lost by age 12 months. This neuronal loss is accompa-
nied by increased markers of oxidative stress and by
increased microglial activation. These changes were
largely prevented by long-term oral administration of
Although EAAC1 is expressed by all CNS neu-
rons,10,11results presented here and previously indicate
that EAAC1 expression is especially dense on SNc dopa-
minergic neurons.11,33This increased expression may
reflect a high basal requirement for glutathione synthesis
in these neurons in response to an intrinsically elevated
rate of oxidant production.8,38Consistent with this idea,
pharmacological inhibition of EAAC1 has been reported
to produce glutathione loss and subsequent cell death
selectively in the dopaminergic neurons of rat and mouse
midbrain.33Similarly, a transgenic mouse constructed by
Chinta and colleagues,39in which glutathione synthesis
is impaired incatecholaminergic
increased protein nitrosylation, reduced mitochondrial
FIGURE 6: N-acetylcysteine improves survival of SNc dopaminergic neurons in EAAC12/2mice. (A) Images prepared as in
Figure 1, with dopaminergic neurons stained green (anti-TH) and neuronal nuclei stained red (anti-NeuN). Neuronal loss in the
SNc is reduced in mice treated with N-acetylcysteine (NAC). Bars 5 200lm. (B) Cell counts of SNc dopaminergic neurons in
EAAC12/2mice with and without NAC treatment. **p < 0.01; n 5 5–6. Note that the cell count data for EAAC12/2mice
without NAC treatment are the same as in Figure 1, reshown here to facilitate comparisons. (C) Sections are stained for
nitrotyrosine (nTyr, green) as a marker of oxidative stress, and for tyrosine hydroxylase (TH, blue) to identify dopaminergic
neurons. Bars 5 50lm. (D) Quantified data show that the nTyr signal localized to dopaminergic neurons is reduced in the
EAAC12/2mice treated with NAC. **p < 0.01; n 5 5–7.
FIGURE 7: N-acetyl-cysteine preserves motor function in
EAAC12/2mice. EAAC12/2and wild-type (WT) mice were
continuously treated with NAC-supplemented water (NAC)
or normal water, and motor agility was evaluated by the
pole test at ages 9 and 12 months. Y-axis shows the mean
time required for mice to invert position and climb down
the vertical pole. *p < 0.05; n 5 7–10.
Berman et al: NAC Prevents SNc Neuronal Loss in EAAC1–/–Mice
March 2011 517
complex 1 activity, and a modest degree of dopaminergic
cell loss. Together, these findings suggest a key role for
EAAC1 in dopaminergic neuronal glutathione metabo-
lism and a contributory role for glutathione depletion in
dopaminergic neuronal death.
Many animal models used to replicate histological
features of PD, such as the 6-hydroxydopamine and
MPTP models, generate massive oxidative stress and cell
death over a few days time.38,40,41By contrast, the oxida-
tive stress associated with human PD is low-grade and
chronic, extending over decades. The ultimate cause of
neuronal death in PD remains uncertain, but evidence
suggests that it may result from accumulated nuclear and
mitochondrial DNA mutations, some of which lead to
additional oxidant production.8,42The EAAC1?/?mouse
provides a model of chronic neuronal oxidative stress,
and could thereby provide insights into cell death mecha-
nisms and therapeutic approaches relevant to chronic oxi-
dative stress. The EAAC1?/?mouse does not, however,
provide a good model of the motor deficits in PD; deficits
were found only with the pole test, and only at 12 months
of age. The paucity of motor findings may reflect the rela-
tively modest degree of dopaminergic neuronal loss
(<50%), which permits sprouting from remaining neu-
rons and other compensatory changes.26,31,32Moreover,
oxidative stress and neuronal loss in EAAC1?/?mice also
occurs to some extent in neuronal populations not signifi-
cantly affected by PD,9a factor that further complicates
interpretation of motor and functional abnormalities.
EAAC1 is also capable of transporting glutamate, albeit
with lower affinity than cysteine.43EAAC1 uptake of
glutamate does not contribute to bulk glutamate clearance
from the extracellular space, but loss of glutamate
transport into neurons could have additional effects on
neuronal metabolism and motor function, unrelated to
It is not known whether changes in EAAC1 expres-
sion or activity occur in PD. Like many transporters, the
activity of EAAC1 is highly regulated by translocation to
and from the cell surface, with only a fraction of avail-
able EAAC1 being present on the cell surface at any one
time.45Several signaling pathways have been identified as
having significant influence on EAAC1 cell-surface
expression, including, protein kinase C, platelet-derived
growth factor, syntaxin 1A, cholesterol, and the gluta-
mate transporter associated protein 3-18 (GTRAP3-
18).45–48Of note, GTRAP3-18 has also been shown to
modulate neuronal glutathione concentrations,34suggest-
ing that perturbations of this or other signaling pathways
that influence EAAC1 cell-surface expression could, in
principle, contribute to the neuronal glutathione deple-
tion observed in PD.
Unlike cysteine, NAC can cross the blood-brain
barrier and passively cross cell membranes to provide
substrate for glutathione synthesis.17,18Glutathione is the
dominant reactive thiol in cells, and it is in equilibrium
with other reactive thiols.49The C5-maleimide method
was used here to show that oral NAC can restore reactive
thiol content in EAAC1?/?dopaminergic SNc neurons.
Prior studies show that the effect of NAC on neuronal
C5-maleimide staining requires glutathione synthesis, fur-
ther indicating that glutathione is a major contributor to
A key finding here is that long-term oral administra-
tion of NAC prevents the neuronal death, oxidative stress,
and motor abnormalities otherwise observed in the aged
EAAC1?/?mice. This finding suggests that NAC or simi-
lar compounds could be used to treat disorders in which
neuronal glutathione synthesis does not meet demand.
NAC is currently approved for use in the United States as
a mucolytic, for treatment of acetaminophen overdose,
and to prevent contrast nephropathy. The drug is also cur-
rently under study for treatment of schizophrenia, amyo-
trophic lateral sclerosis, and other disorders.19In addition,
an extensive literature documents salutary effects of NAC
in other settings involving oxidative stress.19It remains to
be established whether glutathione depletion is a primary
cause of oxidative stress in PD, a result of oxidative stress,
or both, but the evidence that the glutathione depletion
can promote dopaminergic neuronal death, coupled with
the ability of NAC to promote glutathione synthesis, pro-
vides a scientific rationale for evaluating glutathione reple-
tion as a treatment approach for PD.
This research was supported by grants from the U.S.
Department of Veterans Affairs (to R.A.S.) and the Mi-
chael J. Fox Foundation for Parkinson’s Research (to
We thank Colleen Hefner for expert technical assis-
tance, and Drs. William Marks and Graham A. Glass for
their critical reviews of the manuscript.
Potential Conflict of Interest
Nothing to report.
1.Bove J, Prou D, Perier C, Przedborski S. Toxin-induced models of
Parkinson’s disease. NeuroRx 2005;2:484–494.
2. Betarbet R, Sherer TB, MacKenzie G, et al. Chronic systemic pesti-
cide exposure reproduces features of Parkinson’s disease. Nat
ANNALS of Neurology
518Volume 69, No. 3
3. Yoritaka A, Hattori N, Uchida K, et al. Immunohistochemical
detection of 4-hydroxynonenal protein adducts in Parkinson
disease. Proc Natl Acad Sci U S A 1996;93:2696–2701.
4. Floor E, Wetzel MG. Increased protein oxidation in human sub-
stantia nigra pars compacta in comparison with basal ganglia and
prefrontal cortex measured with an improved dinitrophenylhydra-
zine assay. J Neurochem 1998;70:268–275.
5. Dexter DT, Sian J, Rose S, et al. Indices of oxidative stress and mi-
tochondrial function in individuals with incidental Lewy body dis-
ease. Ann Neurol 1994;35:38–44.
6. Sian J, Dexter DT, Lees AJ, et al. Alterations in glutathione levels
in Parkinson’s disease and other neurodegenerative disorders
affecting basal ganglia. Ann Neurol 1994;36:348–355.
7.Schober A. Classic toxin-induced animal models of Parkinson’s
disease: 6-OHDA and MPTP. Cell Tissue Res 2004;318:215–224.
8.Thomas B, Beal MF. Parkinson’s disease. Hum Mol Genet 2007;
9. Aoyama K, Suh SW, Hamby AM, et al. Neuronal glutathione defi-
ciency and age-dependent neurodegeneration in the EAAC1 defi-
cient mouse. Nat Neurosci 2006;9:119–126.
10. Rothstein JD, Martin L, Levey AI, et al. Localization of neuronal
and glial glutamate transporters. Neuron 1994;13:713–725.
11. Shashidharan P, Huntley GW, Murray JM, et al. Immunohisto-
chemical localization of the neuron-specific glutamate trans-
porter EAAC1 (EAAT3) in rat brain and spinal cord revealed
bya novel monoclonalantibody.
12.Watabe M, Aoyama K, Nakaki T. Regulation of glutathione
synthesis via interaction between glutamate transport-associated
protein 3–18 (GTRAP3–18) and excitatory amino acid carrier-1
13. Himi T, Ikeda M, Yasuhara T, et al. Role of neuronal glutamate
transporter in the cysteine uptake and intracellular glutathione lev-
els in cultured cortical neurons. J Neural Transm 2003;110:
14.Dringen R. Metabolism and functions of glutathione in brain. Prog
15.Zeevalk GD, Razmpour R, Bernard LP. Glutathione and Parkinson’s
disease: is this the elephant in the room? Biomed Pharmacother.
16.Offen D, Ziv I, Sternin H, et al. Prevention of dopamine-induced
cell death by thiol antioxidants: possible implications for treat-
ment of Parkinson’s disease. Exp Neurol 1996;141:32–39.
17.Farr SA, Poon HF, Dogrukol-Ak D, et al. The antioxidants alpha-
lipoic acid and N-acetylcysteine reverse memory impairment and
brain oxidative stress in aged SAMP8 mice. J Neurochem 2003;
18.Dringen R, Hamprecht B. N-acetylcysteine, but not methionine or
2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the
synthesis of glutathione in cultured neurons derived from embry-
onal rat brain. Neurosci Lett 1999;259:79–82.
19.Dodd S, Dean O, Copolov DL, et al. N-acetylcysteine for antioxi-
dant therapy: pharmacology and clinical utility. Expert Opin Biol
20.Peghini P, Janzen J, Stoffel W. Glutamate transporter EAAC-1-de-
ficient mice develop dicarboxylic aminoaciduria and behavioral
abnormalities but no neurodegeneration. EMBO J 1997;16:
21. Mutant mice and neuroscience,: recommendations concerning
genetic background. Banbury Conference on genetic background
in mice. Neuron 1997;19:755–759.
22.Kauppinen TM, Higashi Y, Suh SW, et al. Zinc triggers microglial
activation. J Neurosci 2008;28:5827–5835.
23.Schmitz C, Hof PR. Design-based stereology in neuroscience.
24.Hof PR, Young WG, Bloom FE, et al. Comparative cytoarchitec-
tonic atlas of the C57BL/6 and 129/Sv mouse brains. 1st ed.
Amsterdam: Elsevier Science B.V., 2000: 275.
25.Kauppinen TM, Suh SW, Berman AE, et al. Inhibition of poly(ADP-
ribose) polymerase suppresses inflammation and promotes recov-
ery after ischemic injury. J Cereb Blood Flow Metab 2009;29:
26.Meredith GE, Kang UJ. Behavioral models of Parkinson’s disease
in rodents: a new look at an old problem. Mov Disord 2006;21:
27.Rozas G, Lopez-Martin E, Guerra MJ, Labandeira-Garcia JL. The
overall rod performance test in the MPTP-treated-mouse model of
Parkinsonism. J Neurosci Methods 1998;83:165–175.
28.Matsuura K, Kabuto H, Makino H, Ogawa N. Pole test is a use-
ful method for evaluating the mouse movement disorder caused
by striatal dopamine depletion. J Neurosci Methods 1997;73:
29. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraf-
fin-embedded tissues: an enhancement method for immunohisto-
chemical staining based on microwave oven heating of tissue
sections. J Histochem Cytochem. 1991;39:741–748.
30.McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive micro-
glia are positive for HLA-DR in the substantia nigra of Parkin-
31.Bezard E, Dovero S, Imbert C, et al. Spontaneous long-term com-
pensatory dopaminergic sprouting in MPTP-treated mice. Synapse
32.Iravani MM, Syed E, Jackson MJ, et al. A modified MPTP treat-
ment regime produces reproducible partial nigrostriatal lesions in
common marmosets. Eur J Neurosci 2005;21:841–854.
33. Nafia I, Re DB, Masmejean F, et al. Preferential vulnerability of
mesencephalic dopamine neurons to glutamate transporter dys-
function. J Neurochem 2008;105:484–496.
34.Watabe M, Aoyama K, Nakaki T. A dominant role of GTRAP3–
18in neuronalglutathione synthesis.
35.Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxy-
nitrite: the good, the bad, and ugly. Am J Physiol 1996;271:
36.Hodara R, Norris EH, Giasson BI, et al. Functional consequences
of alpha-synuclein tyrosine nitration: diminished binding to lipid
vesicles and increased fibril formation. J Biol Chem 2004;279:
37.Duda JE, Lee VM, Trojanowski JQ. Neuropathology of synuclein
aggregates. J Neurosci Res 2000;61:121–127.
38. Andersen JK. Oxidative stress in neurodegeneration: cause or
consequence? Nat Med 2004;10(suppl):S18–S25.
39.Chinta SJ, Kumar MJ, Hsu M, et al. Inducible alterations of gluta-
thione levels in adult dopaminergic midbrain neurons result in ni-
grostriatal degeneration. J Neurosci 2007;27:13997–14006.
40. Melrose HL, Lincoln SJ, Tyndall GM, Farrer MJ. Parkinson’s dis-
ease: a rethink of rodent models. Exp Brain Res 2006;173:
41.Betarbet R, Sherer TB, Greenamyre JT. Animal models of Parkin-
son’s disease. Bioessays 2002;24:308–318.
42. Bender A, Krishnan KJ, Morris CM, et al. High levels of mitochon-
drial DNA deletions in substantia nigra neurons in aging and Par-
kinson disease. Nat Genet 2006;38:515–517.
43. Zerangue N, Kavanaugh MP. Interaction of L-cysteine with a
human excitatory amino acid transporter. J Physiol 1996;493:
Berman et al: NAC Prevents SNc Neuronal Loss in EAAC1–/–Mice
March 2011 519
44. Sepkuty JP, Cohen AS, Eccles C, et al. A neuronal glutamate Download full-text
transporter contributes to neurotransmitter GABA synthesis and
epilepsy. J Neurosci 2002;22:6372–6379.
45.Fournier KM, Gonzalez MI, Robinson MB. Rapid trafficking of the
neuronal glutamate transporter, EAAC1: evidence for distinct traf-
ficking pathways differentially regulated by protein kinase C and pla-
telet-derived growth factor. J Biol Chem 2004;279:34505–34513.
46.Yu YX, Shen L, Xia P, et al. Syntaxin 1A promotes the endocytic
sorting of EAAC1 leading to inhibition of glutamate transport.
J Cell Sci 2006;119:3776–3787.
47. Canolle B, Masmejean F, Melon C, et al. Glial soluble factors
regulate the activity and expression of the neuronal glutamate
transporter EAAC1: implication of cholesterol. J Neurochem 2004;
48. Lin CI, Orlov I, Ruggiero AM, et al. Modulation of the neuronal
glutamate transporter EAAC1 by the interacting protein GTRAP3–
18. Nature 2001;410:84–88.
49.Jones DP, Go YM, Anderson CL, et al. Cysteine/cystine couple is
a newly recognized node in the circuitry for biologic redox
signaling and control. FASEB J. 2004;18:1246–1248.
ANNALS of Neurology
520 Volume 69, No. 3