Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress.
ABSTRACT Mutations of the DJ-1 (PARK7) gene are linked to familial Parkinson's disease. We used gene targeting to generate DJ-1-deficient mice that were viable, fertile, and showed no gross anatomical or neuronal abnormalities. Dopaminergic neuron numbers in the substantia nigra and fiber densities and dopamine levels in the striatum were normal. However, DJ-1-/- mice showed hypolocomotion when subjected to amphetamine challenge and increased striatal denervation and dopaminergic neuron loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. DJ-1-/-embryonic cortical neurons showed increased sensitivity to oxidative, but not nonoxidative, insults. Restoration of DJ-1 expression to DJ-1-/- mice or cells via adenoviral vector delivery mitigated all phenotypes. WT mice that received adenoviral delivery of DJ-1 resisted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine-induced striatal damage, and neurons overexpressing DJ-1 were protected from oxidative stress in vitro. Thus, DJ-1 protects against neuronal oxidative stress, and loss of DJ-1 may lead to Parkinson's disease by conferring hypersensitivity to dopaminergic insults.
- SourceAvailable from: Javier Blesa[Show abstract] [Hide abstract]
ABSTRACT: Parkinson's disease (PD) is a neurodegenerative disorder that affects about 1.5% of the global population over 65 years of age. A hallmark feature of PD is the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and the consequent striatal DA deficiency. Yet, the pathogenesis of PD remains unclear. Despite tremendous growth in recent years in our knowledge of the molecular basis of PD and the molecular pathways of cell death, important questions remain, such as: 1) why are SNc cells especially vulnerable; 2) which mechanisms underlie progressive SNc cell loss; and 3) what do Lewy bodies or α-synuclein reveal about disease progression. Understanding the variable vulnerability of the dopaminergic neurons from the midbrain and the mechanisms whereby pathology becomes widespread are some of the primary objectives of research in PD. Animal models are the best tools to study the pathogenesis of PD. The identification of PD-related genes has led to the development of genetic PD models as an alternative to the classical toxin-based ones, but does the dopaminergic neuronal loss in actual animal models adequately recapitulate that of the human disease? The selection of a particular animal model is very important for the specific goals of the different experiments. In this review, we provide a summary of our current knowledge about the different in vivo models of PD that are used in relation to the vulnerability of the dopaminergic neurons in the midbrain in the pathogenesis of PD.Frontiers in Neuroanatomy 12/2014; 8. · 4.18 Impact Factor
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ABSTRACT: Mitochondria are small organelles that produce the majority of cellular energy as ATP. Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD), and rare familial forms of PD provide valuable insight into the pathogenic mechanism underlying mitochondrial impairment, even though the majority of PD cases are sporadic. The regulation of mitochondria is crucial for the maintenance of energy-demanding neuronal functions in the brain. Mitochondrial biogenesis and mitophagic degradation are the major regulatory pathways that preserve optimal mitochondrial content, structure and function. In this mini-review, we provide an overview of the mitochondrial quality control mechanisms, emphasizing regulatory molecules in mitophagy and biogenesis that specifically interact with the protein products of three major recessive familial PD genes, PINK1, Parkin and DJ-1.Experimental neurobiology. 12/2014; 23(4):345-51.
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ABSTRACT: Aging is associated with the accumulation of various deleterious changes in cells. According to the free radical and mitochondrial theory of aging, mitochondria initiate most of the deleterious changes in aging and govern life span. The failure of mitochondrial reduction-oxidation (redox) homeostasis and the formation of excessive free radicals are tightly linked to dysregulation in the Renin Angiotensin System (RAS). A main rate-controlling step in RAS is renin, an enzyme that hydrolyzes angiotensinogen to generate angiotensin I. Angiotensin I is further converted to Angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II binds with equal affinity to two main angiotensin receptors-type 1 (AT1R) and type 2 (AT2R). The binding of Ang II to AT1R activates NADPH oxidase, which leads to increased generation of cytoplasmic reactive oxygen species (ROS). This Ang II-AT1R-NADPH-ROS signal triggers the opening of mitochondrial KATP channels and mitochondrial ROS production in a positive feedback loop. Furthermore, RAS has been implicated in the decrease of many of ROS scavenging enzymes, thereby leading to detrimental levels of free radicals in the cell. AT2R is less understood, but evidence supports an anti-oxidative and mitochondria-protective function for AT2R. The overlap between age related changes in RAS and mitochondria, and the consequences of this overlap on age-related diseases are quite complex. RAS dysregulation has been implicated in many pathological conditions due to its contribution to mitochondrial dysfunction. Decreased age-related, renal and cardiac mitochondrial dysfunction was seen in patients treated with angiotensin receptor blockers. The aim of this review is to: (a) report the most recent information elucidating the role of RAS in mitochondrial redox hemostasis and (b) discuss the effect of age-related activation of RAS on generation of free radicals.Frontiers in Physiology 11/2014; 5:439.
Hypersensitivity of DJ-1-deficient mice to
and oxidative stress
Raymond H. Kim*†, Patrice D. Smith†‡, Hossein Aleyasin‡, Shawn Hayley§, Matthew P. Mount‡, Scott Pownall¶,
Andrew Wakeham*, Annick J. You-Ten*, Suneil K. Kalia?, Patrick Horne**, David Westaway**, Andres M. Lozano?,
Hymie Anisman§, David S. Park‡††‡‡, and Tak W. Mak*††‡‡
*Campbell Family Institute for Breast Cancer Research, Advanced Medical Discovery Institute, Ontario Cancer Institute and Department of Medical
Biophysics, University of Toronto, Toronto, ON, Canada M5G 2C1;‡Neuroscience Research Group, Ottawa Health Research Institute, University of Ottawa,
Ottawa, ON, Canada K1H 8M5;§Institute for Neuroscience, Carleton University, Ottawa, ON, Canada K1H 6N5;¶enGene Incorporated, Vancouver, BC,
Canada V6T 1Z3;?Division of Applied and Interventional Research, Toronto Western Hospital Research Institute, Toronto, ON, Canada M5T 2S8; and
**Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada M5S 3H2
Contributed by Tak W. Mak, February 16, 2005
Mutations of the DJ-1 (PARK7) gene are linked to familial Parkin-
son’s disease. We used gene targeting to generate DJ-1-deficient
mice that were viable, fertile, and showed no gross anatomical or
neuronal abnormalities. Dopaminergic neuron numbers in the
substantia nigra and fiber densities and dopamine levels in the
striatum were normal. However, DJ-1??? mice showed hypolo-
comotion when subjected to amphetamine challenge and in-
by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. DJ-1??? em-
bryonic cortical neurons showed increased sensitivity to oxidative,
but not nonoxidative, insults. Restoration of DJ-1 expression to
DJ-1??? mice or cells via adenoviral vector delivery mitigated
all phenotypes. WT mice that received adenoviral delivery of
DJ-1 resisted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine-induced
striatal damage, and neurons overexpressing DJ-1 were protected
from oxidative stress in vitro. Thus, DJ-1 protects against neuronal
oxidative stress, and loss of DJ-1 may lead to Parkinson’s disease
by conferring hypersensitivity to dopaminergic insults.
(1). The cause of PD remains unknown, but epidemiological and
genetic studies have suggested that the observed loss of dopami-
nergic neurons in PD is due to defects in common intracellular
signaling pathways (2). Genes linked to familial PD include
?-synuclein (3), Parkin (4), UCH-L1 (5), PINK1 (6), and dardarin
(7). Proteins encoded by these genes are thought to be involved in
protein aggregation and proteasome function, processes which,
when disrupted in model systems, can also result in noninherited
forms of PD (8). Recently, loss-of-function mutations in the DJ-1
locus were found in families with autosomal recessive early-onset
PD (9). Additional studies have confirmed other DJ-1 mutations in
various PD cohorts (10). DJ-1 was initially cloned as a putative
oncogene (11) and as part of an RNA-binding complex (12). DJ-1
is highly expressed by normal astrocytes (13) and has been impli-
cated in fertilization (14) and tumorigenesis (15, 16). Studies of the
crystal structure of DJ-1 (17) suggest that a particular DJ-1 muta-
tion (L166P) reduces DJ-1 protein stability (18–20), resulting in
degradation through the ubiquitin–proteasome system (21, 22).
However, the physiological function of DJ-1 remains largely un-
Motor impairments in PD patients result from inhibition of the
nigrostriatal motor pathway. This inhibition is due to the loss of
dopaminergic neurons in the substantia nigra pars compacta (SNc)
(8). The cause of the dopaminergic neuron loss remains unknown,
but oxidative stress leading to apoptotic neuronal death has been
an effort to reproduce oxidative stress leading to neuronal loss in
the SNc. Of these, administration of the well characterized meper-
arkinson’s disease (PD) is a neurodegenerative disorder char-
acterized by tremor, rigidity, akinesia, and postural instability
idine analogue 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine
(MPTP) results in pathology most similar to PD (24). When taken
up by dopaminergic neurons, MPP? (the active metabolite of
MPTP) inhibits mitochondrial complex I and thus impairs respi-
ration, leading to superoxide formation (23). Although in vitro
studies have suggested that DJ-1 can protect cultured neuronal cell
lines against the effects of oxidative stress (25), the in vivo role of
DJ-1 has yet to be determined. To investigate the physiological
function of DJ-1 and to examine the effect of DJ-1 deficiency in
vivo, we have characterized gene-targeted DJ-1 knockout (DJ-
1???) mice. We demonstrate that loss of DJ-1 exacerbates oxi-
dative stress-induced cell death in primary cortical and dopami-
protect WT neurons from oxidative stress. We also show that
susceptibility to MPTP-induced striatal fiber and nigral neuronal
loss is increased in DJ-1??? mice. In WT mice, adenoviral-
mediated overexpression of DJ-1 blocks MPTP-induced neuronal
Our results point to a physiological role for DJ-1 in the protection
of neurons against oxidative stress and environmental neurotoxins.
Generation and Genotyping of DJ-1-Deficient Mice. The genomic
murine DJ-1 gene was isolated from a 129?Sv library and used to
generate a targeting construct in which DJ-1 exons 3–5 were
exon of DJ-1 was modified to contain a premature stop codon,
resulting in a transcript corresponding to the first eight amino acids
of the DJ-1 cDNA. Targeting constructs were electroporated into
E14K embryonic stem cells (129?Ola). G418-resistant colonies
were screened by PCR (forward primer, TGC TGA AAC TCT
TCA T). PCR-positive colonies were confirmed by Southern blot-
ting of EcoRI-digested genomic DNA by using flanking DJ-1
genomic and neomycin-specific probes. Successful homologous
recombinants were injected into day 3.5 C57BL?6 blastocytes to
generate chimeric mice. Chimeric males were crossed with
C57BL?6 females to achieve germ-line transmission identified by
coat color and confirmed by Southern blotting of tail genomic
Freely available online through the PNAS open access option.
Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine; MPP?, active me-
hydroxylase; DAT, dopamine transporter protein.
†R.H.K. and P.D.S. contributed equally to this work.
††D.S.P. and T.W.M. contributed equally to this work.
‡‡To whom correspondence may be addressed. E-mail: email@example.com or tmak@
© 2005 by The National Academy of Sciences of the USA
April 5, 2005 ?
vol. 102 ?
no. 14 ?
DNA. F1 progeny were backcrossed for seven generations to
C57BL?6 mice, and heterozygotes were intercrossed to generate
mice homozygous for the targeted DJ-1 allele. Genotypes of
animals were verified by using PCR (WT DJ-1 forward primer,
TGC TGA AAC TCT GCC ATG TGA ACC; WT DJ-1 reverse
primer, CCT GCT TGC CGA ATA TCA T; and Neo, AGG TGA
CAC TGC CAG TTG CTA GTC). PCR conditions were 95°C for
30 sec, 64°C for 30 sec, and 72°C for 1 min (40 cycles).
Generation of Anti-DJ-1 Antibody. Rabbit antiserum was raised
purified by preadsorption on Trx-coupled CNBr-Sepharose 4B
followed by affinity purification on GST-DJ-1 fusion protein cou-
pled to CNBr-Sepharose 4B. Low-affinity antibody was eluted in a
in this study was eluted from the affinity column with 0.1 M glycine
Generation of DJ-1 Adenoviruses. Adenovirus vectors expressing
DJ-1 were generated by subcloning WT DJ-1 cDNA or L166P
mutant DJ-1 cDNA into pAdTRACK-CMV (26) in which the
expression of GFP and DJ-1 is driven by separate cytomegalovirus
Neuronal Cultures and Stimuli. Cortical neurons were cultured as
described (27) from day 14–15 mouse embryos either of the CD1
strain (Charles River Laboratories) or from DJ-1???, DJ-1???,
or DJ-1??? animals generated by knockout breeding. On day 1–2
after the initial plating, neurons were cultured in serum-free
or staurosporine (2 ?M). Numbers of viable neurons were evalu-
ated by lysis of cultures followed by the counting of intact nuclei as
For overexpression studies involving H2O2treatment, cortical
neurons were exposed to recombinant adenovirus expressing GFP
only, GFP plus DJ-1 cDNA, or GFP plus DJ-1 L166P mutant
with 30 ?M H2O2and then fixed. Infected cells were identified by
GFP fluorescence, and nuclear integrity was assessed by Hoechst
as the percentage of live GFP-positive neurons over the total
number of neurons expressing GFP.
For experiments involving mesencephalic neurons, midbrain
cultures were harvested from day 13–14 embryos as described (29).
Cells were incubated with anti-tyrosine hydroxylase (TH) antibody
(ImmunoStar, Hudson, WI; 1:10,000) as the primary antibody and
Cy3-linked anti-mouse antibody (1:300, The Jackson Laboratory)
as the secondary antibody. Nuclei were stained with Hoechst dye.
TH-positive (TH?) cells were visualized by fluorescence micros-
copy, and neuronal viability was evaluated by nuclear morphology.
the treated culture compared with untreated controls.
MPTP Treatment and Adenoviral Gene Delivery in Vivo. Littermate
all MPTP experiments. On 5 consecutive days, mice received i.p.
injections of either MPTP?HCl (Sigma; 25 mg of free base per kg
of body weight per day) or an equivalent volume of 0.9% saline. At
14 days post-MPTP treatment, all mice were killed and their
neurons analyzed. For mice pretreated with adenoviral vectors,
(30). The adenovirus was delivered into the striatum 7 days before
the initiation of MPTP treatment. All animal experiments con-
formed to the guidelines set by the Canadian Council for the Use
and Care of Animals in Research and the Canadian Institutes of
Health Research and were conducted with the approval of the
University of Ottawa and Ontario Cancer Institute Animal Care
Immunohistochemistry. Mice were killed and brains harvested as
described (31). Free-floating sections were incubated with primary
anti-TH antibody (1:10,000, ImmunoStar), anti-DJ-1 antibody
(1:1,000), a biotinylated secondary antibody (1:200, Jackson Im-
antibody (1:200, Amersham Pharmacia Bioscience).
Neuronal Loss Assessment. After immunohistochemical analysis,
TH?neurons were counted as described (32). Loss of TH?or
cresyl violet-stained cells in the nigral region was used as an
index of dopaminergic cell loss after MPTP treatment. For
adenoviral experiments, TH?cell counts were performed as
described (30) at the level of medial terminal nucleus, the region
in which adenovirus-mediated gene expression is highest (30).
For analyses of TH and dopamine transporter protein (DAT)
intensity in the striatum, sections were analyzed by densitometry
by using NORTHERN ECLIPSE IMAGE software, as described (30).
HPLC and MPP?Analysis. Analyses of dopamine, dopamine metab-
olites, and MPP?were carried out as described (33).
Behavioral Analyses. Novel environment. Walled 30 ? 30-inch square
arenas were used for open field testing of mice by using a video
camera and analysis software, as described (30).
Motor impairments. Assessment of the ability of mice to descend and
cross poles (pole test) was carried out as described (34).
Somatosensory. The adhesive removal test was used to assess so-
matosensory ability and was carried out as described (35).
Home cage. Animal behavior was assessed by using a home-cage
mouse-monitoring system that utilizes the beam-break sensor ap-
paratus (MicroMax, Accuscan, Columbus, OH). Animals were
monitored for 24 h before MPTP treatment or starting on day 13
after the initiation of MPTP treatment. To assess ‘‘dopamine-
related’’ behavioral output, mice were monitored for 1 hr after
amphetamine challenge (2 mg?kg) (30).
Generation of DJ-1 Knockout Mice. We generated DJ-1-deficient
mice by gene-targeting (Fig. 6A, which is published as supporting
DJ-1 locus by Southern blotting (Fig. 6B). Northern blotting of
RNA from DJ-1??? mouse embryonic fibroblasts (Fig. 6C) and
confirmed the lack of endogenous DJ-1 RNA and protein expres-
sion. DJ-1??? and DJ-1??? mice were born at the expected
Mendelian frequencies, were viable and fertile, and showed no
gross anatomical or neuronal abnormalities.
DJ-1 Protects Primary Neurons Against Cell Death Induced by Oxida-
tive Stress. To determine whether DJ-1 could protect primary
neurons derived from the brains of DJ-1???, DJ-1???, and
DJ-1??? embryos to oxidative stress in the form of H2O2. DJ-
was observed in DJ-1??? neurons, suggesting a gene-dosage
effect. To determine whether overexpression of DJ-1 could protect
primary DJ-1??? neurons from oxidative death, we constructed
adenovirus vectors expressing either GFP alone (control) or GFP
plus WT DJ-1 (Fig. 1B). Primary DJ-1??? cortical neurons were
infected with these vectors and treated with H2O2. Overexpression
of WT DJ-1 protected DJ-1??? neurons against H2O2-induced
apoptosis (Fig. 1C). However, this protection was not observed if
DJ-1??? neurons were infected with an adenovirus vector ex-
pressing GFP alone or GFP plus the mutated L166P DJ-1 protein
www.pnas.org?cgi?doi?10.1073?pnas.0501282102Kim et al.
(Fig. 1C). Indeed, a slight increase in cell death was observed in
may act as a dominant negative inhibitor of WT DJ-1. When we
overexpressed WT DJ-1 in DJ-1??? cortical neurons, the hyper-
sensitivity of the mutant cells to H2O2 was reduced (Fig. 1D),
confirming that DJ-1 protects primary neurons from oxidative
The pesticide rotenone is another oxidative stressor thought to
promote neuronal death in PD (36). Rotenone inhibits mitochon-
drial complex I and increases reactive oxygen species (37). We
treated DJ-1??? mesencephalic dopaminergic neurons (which
stain positively for TH?) with rotenone and analyzed cell death.
The survival of rotenone-treated DJ-1??? TH?neurons was
decreased by 30% compared with rotenone-treated DJ-1???
neurons (Fig. 1E).
To investigate whether DJ-1 protects against nonoxidative in-
sults, cortical neurons from DJ-1???, DJ-1???, and DJ-1???
embryos were treated with various doses of camptothecin, a topo-
isomerase I inhibitor. No statistically significant differences in cell
death were observed among the genotypes (Fig. 1F). Neither were
DJ-1??? neurons more susceptible than controls to treatment
with the protein kinase inhibitor staurosporine (Fig. 1G).
Behavioral Defects in DJ-1??? Mice. To determine whether DJ-
1??? mice exhibited gross motor behavior abnormalities resem-
bling those in PD patients, we carried out open field locomotion
analyses over a 24-h period using automated beam break analyses
in a home-cage environment. However, no statistically significant
differences in spontaneous locomotion could be detected among
DJ-1???, DJ-1???, and DJ-1??? mice (8 weeks old; C57BL?6)
background (data not shown). Additional behavior analyses, in-
cluding the pole (34), novel environment (30), and somatosensory
induced by oxidative, but not nonoxida-
tive, stress. Survival determinations for
each of the following experiments are de-
scribed in Experimental Procedures. (A) In-
DJ-1. Cortical neurons from DJ-1???, DJ-
1???, and DJ-1??? embryos were treated
for 3 h with 30 ?M H2O2. (B) Immunofluo-
rescence analysis of DJ-1??? embryonic
cortical neurons infected with an adenovi-
plus DJ-1. Hoechst nuclear staining of the
same culture is also shown. (C) Protective
effect of DJ-1 overexpression in DJ-1???
cortical neurons. DJ-1??? neurons in-
WT DJ-1 protein, but not GFP plus mutated
cortical neurons. DJ-1??? neurons in-
WT DJ-1 showed increased survival after
H2O2exposure. (E) Increased sensitivity of
DJ-1??? mesencephalic dopaminergic
neurons (TH?) to other oxidative insults.
DJ-1??? cortical neurons were exposed to
10 nM rotenone for 24 h. (F and G) Lack of
sensitivity of DJ-1??? cortical neurons
doses of camptothecin (Fig. 1F) or 2 ?M
staurosporine (Fig. 1G) were applied to DJ-
1???, DJ-1???, and DJ-1??? neurons for
16 h. For A and C–G, each data point is the
mean ? SEM of three to five independent
cultures, where*denotes a significance
level of P ? 0.05.
DJ-1 protects against cell death
Kim et al.PNAS ?
April 5, 2005 ?
vol. 102 ?
no. 14 ?
tests (35), were performed on aged DJ-1??? mice, but no anom-
mice tended to display a slightly lower average locomotor activity
The locomotor activity of MPTP-treated DJ-1??? and DJ-1???
mice was reduced by 50% compared with saline-treated controls
mice, MPTP-treated DJ-1??? mice showed a statistically signifi-
cant decrease in total activity (Fig. 2A Right). In a parallel exper-
iment, we challenged DJ-1??? and DJ-1??? mice with amphet-
amine, which leads to dopamine release (38) and hyperlocomotion
in WT mice (39). DJ-1??? mice exhibited a significant depression
in amphetamine-induced locomotor activity compared with con-
trols (Fig. 2B). Thus, although unchallenged locomotor behavior is
normal in the absence of DJ-1, a mild deficit exists that is revealed
upon challenge of the dopaminergic system.
Loss of DJ-1 Confers Susceptibility to MPTP-Induced Neuronal Death.
We next histologically examined the effects of chronic MPTP
neurotoxicity on DJ-1??? mice. Saline-treated DJ-1??? and
DJ-1??? mice showed no significant differences in TH?neurons
(Fig. 3 A and B). However, at 14 days post-MPTP treatment,
neuron numbers compared with saline-treated controls (Fig. 3 A
and C). Strikingly, DJ-1??? mice showed a much greater relative
decrease in the number of viable TH?SNc neurons after MPTP
treatment (Fig. 3 B and D), suggesting that loss of DJ-1 sensitizes
SNc neurons to MPTP-induced apoptosis. Quantitation of TH?
neuron numbers in MPTP-treated DJ-1??? and DJ-1??? mice
confirmed the histological findings (Fig. 3E). Similar results were
obtained by using cresyl violet staining (40) as an independent
marker of neuronal survival (Fig. 3F).
DJ-1??? Mice Show Increased Striatal Denervation After MPTP
Treatment. We confirmed that MPTP induces a greater loss of
dopaminergic neurons in the absence of DJ-1 by examining dopa-
minergic terminal fiber density and dopamine levels in the striatal
similar TH staining in the striatum (Fig. 4 A and B). Although
MPTP-treated DJ-1??? mice displayed the expected moderate
depletion of dopaminergic fibers (Fig. 4 A and C), loss of DJ-1
exacerbated this effect (Fig. 4 B and D). Adjacent tissue sections
enhanced loss of striatal fiber density was confirmed by densito-
metric analysis of the TH- (Fig. 4I) and DAT-stained (Fig. 4J)
sections. HPLC analyses revealed a significant reduction in striatal
dopamine in all MPTP-treated mice compared with saline-treated
controls (Fig. 4K). However, the relative loss of dopamine in
MPTP-treated DJ-1??? mice exceeded that in MPTP-treated
DJ-1??? mice (Fig. 4K), consistent with our histological data. No
significant differences in dopamine metabolites were detected
between DJ-1??? and DJ-1??? mice (data not shown). Impor-
tantly, the increased MPTP sensitivity of DJ-1??? mice was not
due to increased production of MPP? in either the SNc (Fig. 4L)
or the striatum (data not shown). Loss of DJ-1 thus increases
susceptibility to dopaminergic neuron degeneration in vivo, which
in turn leads to decreased striatal dopamine.
over a 24-h period in either unchallenged animals (basal) or animals that had
been challenged 14 days earlier with MPTP (30 mg?kg per day for 5 days) (n ?
6–10 animals per group). (B) Open-field activity measured for 1 h in DJ-1???
and DJ-1??? mice (n ? 4 animals?group) challenged with 2 mg?kg amphet-
amine. Total activity was measured in arbitrary units and plotted as the
mean ? SEM, where*denotes a significance level of P ? 0.05.
Minor behavioral defects are revealed in DJ-1??? mice only upon
MPTP or saline (control) as for Fig. 2, and
terminal nucleus were prepared 14 days
later. Sections were immunostained to de-
tect TH?neurons. Shown are the SNc of
saline-treated DJ-1??? (A) and DJ-1???
(B) mice and SNc of MPTP-treated DJ-1???
(C) and DJ-1??? (D) mice. (E and F) Quan-
tification of SNc neurons in the mice in A–D
using either TH staining (E) or cresyl violet
mean ? SEM (n ? 5–7 animals per group),
where*and**denote significance levels
(ANOVA) of P ? 0.05 and P ? 0.01,
Loss of DJ-1 confers susceptibility
www.pnas.org?cgi?doi?10.1073?pnas.0501282102Kim et al.
MPTP-Induced Neuronal Loss Is Rescued by Adenoviral Expression of
DJ-1. To investigate whether DJ-1 could protect against oxidative
insults in vivo, we engineered the restoration of DJ-1 expression in
the SNc of DJ-1??? mice using retrograde transportation of
adenoviruses (31) expressing either DJ-1 or LacZ (control). Viral
vectors were injected into one hemisphere of the striatum of
DJ-1??? mice. Virally introduced DJ-1 (but not LacZ) led to
substantial DJ-1 expression in the SNc of the ipsilateral side of
injection (Fig. 5 A and B).
DJ-1??? and?or DJ-1??? mice from MPTP-induced neurode-
generation. Viral vectors expressing either DJ-1 or LacZ were
unilaterally injected into the striatum of DJ-1??? and DJ-1???
contralateral side of the striatum of MPTP-treated mice served as
the MPTP-treated uninjected control. Expression of DJ-1 in DJ-
1??? (Fig. 5 C, D, and G), and DJ-1??? (Fig. 5 E–G) mice
prevented much of the neuronal loss induced by MPTP. In saline-
MPTP or saline as for Fig. 2, and striatal
sections were prepared 14 days later. Sec-
tions were immunostained to detect either
TH?neurons (A–D) or DAT?neurons (E–H).
DJ-1??? mice were treated with saline (A
and E) or MPTP (C and G), and DJ-1???
mice were treated with saline (B and F) or
MPTP (D and H). (I and J) Quantification of
striatal fiber density (I; TH staining) and
nerve density (J; DAT staining) of mice in
A–H. HPLC determination of striatal dopa-
mine (DA) levels (K) and nigral MPP? levels
(L) in DJ-1 control (??? or ???) and DJ-
1??? mice. All animals were treated with
either MPTP or saline as for Fig. 2 and eval-
uated on day 14. Data in I–L are presented
as mean ? SEM (n ? 4–8 animals?group),
where*and**denote significance levels
(ANOVA) of P ? 0.05 and P ? 0.01,
DJ-1??? mice show increased stri-
cued by adenoviral expression of DJ-1. (A
and B) DJ-1??? mice were injected unilat-
erally in the SNc with adenoviral vectors
expressing either DJ-1 or LacZ (control).
Sections of the SNc at the medial terminal
nucleus were immunostained with anti-
DJ-1 antibody to detect adenoviral-medi-
ated DJ-1 expression. (A) DJ-1 expression is
not visible in the contralateral (uninjected)
hemisphere of DJ-1??? SNc. (B) Upon in-
jection of vector expressing WT DJ-1, DJ-1
expression is restored to the ipsilateral
hemisphere of DJ-1??? SNc. (C–F) DJ-
laterally in the SNc with adenoviral vectors
expressing either DJ-1 or LacZ (control; not
with MPTP as for Fig. 2. SNc sections were
on the contralateral (no DJ-1 injection) side of the SNc of an MPTP-treated DJ-1??? mouse when compared with (D) the ipsilateral (DJ-1-injected) side of the
animals to oxidative stress. (F) Overexpression of DJ-1 in the ipsilateral side of the DJ-1??? SNc attenuates this neuronal loss. (G) Quantification of numbers of
TH?neurons in the contralateral and ipsilateral SNc hemispheres of all animals in the groups represented in A–F. Data in G are presented as mean ? SEM (n ?
4–5 animals per group), where**denotes a significance level (ANOVA) of P ? 0.01.
MPTP-induced neuronal loss is res-
Kim et al. PNAS ?
April 5, 2005 ?
vol. 102 ?
no. 14 ?
treated DJ-1??? mice, expression of DJ-1 did not alter SNc
neuron numbers compared with LacZ-expressing controls (Fig.
5G). As expected, MPTP treatment of LacZ-expressing DJ-1???
mice induced a 30–40% decrease in TH?neuron numbers in both
the ipsilateral and contralateral hemispheres (Fig. 5G). However,
DJ-1 expression in the ipsilateral SNc of MPTP-treated DJ-1???
or DJ-1??? mice resulted in a statistically significant rescue of
TH?neuron numbers (Fig. 5G). These data show that oxidative
stress resulting from MPTP-mediated inhibition of mitochondrial
complex I can be mitigated by overexpression of DJ-1. Thus, the
hypersensitivity to MPTP observed in DJ-1??? mice is a direct
result of their DJ-1 deficiency.
In this study, we characterized gene-targeted mice lacking DJ-1, a
gene associated with familial PD. Mice deficient in DJ-1 are viable
and fertile and produce viable offspring at the expected Mendelian
frequencies. These data preclude a role for DJ-1 in embryogenesis
or fertility. Rather, our results strongly suggest that a key physio-
logical role of DJ-1 is to protect dopaminergic neurons in the SNc
from oxidative stress. Although DJ-1 has been previously shown to
results demonstrate this protective function in primary neurons in
vivo. Furthermore, our data indicate that loss of DJ-1 function
increases the sensitivity of primary neurons to oxidative stress and
thus may promote neurodegeneration and PD development.
DJ-1 is highly expressed in neuronal and nonneuronal cells, and
it has been proposed that DJ-1 might exert its neuroprotective
effects by influencing the interaction of neurons and glia (13).
neurons is sufficient to confer hypersensitivity to oxidative stress.
Furthermore, these effects can be rescued by expression of the
exogenous WT DJ-1 protein but not of the mutated L166P DJ-1
acts as a dimer, and that the L166P mutation inhibits dimer
formation by breaking the C-terminal helix (17). When expressed
in WT primary neurons, L166P DJ-1 may act as a dominant-
negative inhibitor of DJ-1 dimerization such that the WT DJ-1
protein is no longer able to exert its antioxidative effect (41). In PD
patients, L166P expression in neurons appears to represent a
loss-of-function mutation (9). When we specifically examined the
the environmental toxin rotenone revealed a heightened sensitivity
in the absence of DJ-1. We also showed that DJ-1 deficiency does
not increase susceptibility to neuronal death induced by nonoxida-
tive stimuli, further supporting the hypothesis that neuronal death
in PD patients is specifically due to oxidative stress.
DJ-1??? mice do not display any gross neuronal abnormalities
or motor deficits. Furthermore, untreated DJ-1??? mice do not
exhibit alterations in: (i) the number of TH?neurons in the SNc,
target striatal region, or (iii) striatal dopamine levels. Only slight
motor deficits are seen when mutant mice are treated with am-
phetamine. These findings indicate that loss of DJ-1 alone is not
a heightened tendency to develop PD-like pathology after MPTP
treatment. MPTP reproduces PD pathology in the SNc of both
humans and mice by affecting the oxidative balance in dopaminer-
gic neurons (24). We found that the striatal denervation in MPTP-
treated DJ-1??? mice was highly reminiscent of the SNc neuronal
loss observed in human PD patients. Future study of these mutant
animals may reveal additional deficits and should allow an exam-
ination of how the dose of an oxidative agent affects susceptibility
to neurodegeneration. Our DJ-1??? mice thus represent a valu-
able model in which to examine the molecular mechanisms under-
D.S.P. was supported by the Canadian Institutes of Health Research
(CIHR), the Parkinson’s Society of Canada, the Parkinson’s Disease
Foundation, the Michael J. Fox Foundation, and the Canadian Stroke
Network. D.S.P. is a CIHR scholar, and H.A. and S.H. are Canada
Research Chairs in Neuroscience. R.H.K. and S.K.K. are recipients of
M.D.?Ph.D. scholarships from the CIHR. R.H.K. is also supported by
the Frank Fletcher Memorial Fund, the David Rae Scholarship, and the
Paul Starita Fellowship.
1. Lang, A. E. & Lozano, A. M. (1998) N. Engl. J. Med. 339, 1044–1053.
2. Dawson, T. M. & Dawson, V. L. (2003) Science 302, 819–822.
3. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B.,
Root, H., Rubenstein, J., Boyer, R., et al. (1997) Science 276, 2045–2047.
4. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S.,
Yokochi, M., Mizuno, Y. & Shimizu, N. (1998) Nature 392, 605–608.
5. Leroy, E., Boyer, R., Auburger, G., Leube, B., Ulm, G., Mezey, E., Harta, G., Brownstein,
M. J., Jonnalagada, S., Chernova, T., et al. (1998) Nature 395, 451–452.
6. Valente, E. M., Abou-Sleiman, P. M., Caputo, V., Muqit, M. M., Harvey, K., Gispert, S., Ali,
Z., Del Turco, D., Bentivoglio, A. R., Healy, D. G., et al. (2004) Science 304, 1158–1160.
7. Paisan-Ruiz, C., Jain, S., Evans, E. W., Gilks, W. P., Simon, J., van der Brug, M., de Munain,
A. L., Aparicio, S., Gil, A. M., Khan, N., et al. (2004) Neuron 44, 595–600.
8. Chung, K. K., Dawson, V. L. & Dawson, T. M. (2003) J. Neurol. 250, 15–24.
9. Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., Dekker,
M. C., Squitieri, F., Ibanez, P., Joosse, M., et al. (2003) Science 299, 256–259.
10. Tan, E. K., Tan, C., Zhao, Y., Yew, K., Shen, H., Chandran, V. R., Teoh, M. L., Yih, Y.,
Pavanni, R., Wong, M. C., et al. (2004) Neurosci. Lett. 367, 109–112.
11. Nagakubo, D., Taira, T., Kitaura, H., Ikeda, M., Tamai, K., Iguchi-Ariga, S. M. & Ariga, H.
(1997) Biochem. Biophys. Res. Commun. 231, 509–513.
12. Hod, Y., Pentyala, S. N., Whyard, T. C. & El-Maghrabi, M. R. (1999) J. Cell. Biochem. 72,
Pittman, A. M., Lashley, T., Canet-Aviles, R., Miller, D. W., et al. (2004) Brain 127, 420–430.
14. Takahashi, K., Taira, T., Niki, T., Seino, C., Iguchi-Ariga, S. M. & Ariga, H. (2001) J. Biol.
Chem. 276, 37556–37563.
15. Le Naour, F., Misek, D. E., Krause, M. C., Deneux, L., Giordano, T. J., Scholl, S. & Hanash,
S. M. (2001) Clin. Cancer Res. 7, 3328–3335.
16. MacKeigan, J. P., Clements, C. M., Lich, J. D., Pope, R. M., Hod, Y. & Ting, J. P. (2003)
Cancer Res. 63, 6928–6934.
17. Tao, X. & Tong, L. (2003) J. Biol. Chem. 278, 31372–31379.
18. Olzmann, J. A., Brown, K., Wilkinson, K. D., Rees, H. D., Huai, Q., Ke, H., Levey, A. I.,
Li, L. & Chin, L. S. (2004) J. Biol. Chem. 279, 8506–8515.
19. Moore, D. J., Zhang, L., Dawson, T. M. & Dawson, V. L. (2003) J. Neurochem. 87,
A., Heutink, P. & Rizzu, P. (2003) Hum. Mol. Genet. 12, 2807–2816.
21. Miller, D. W., Ahmad, R., Hague, S., Baptista, M. J., Canet-Aviles, R., McLendon, C.,
Carter, D. M., Zhu, P. P., Stadler, J., Chandran, J., et al. (2003) J. Biol. Chem. 278,
22. Gorner, K., Holtorf, E., Odoy, S., Nuscher, B., Yamamoto, A., Regula, J. T., Beyer, K.,
Haass, C. & Kahle, P. J. (2004) J. Biol. Chem. 279, 6943–6951.
23. Jenner, P. (2003) Ann. Neurol. 53, S26–S38.
24. Przedborski, S. & Vila, M. (2003) Ann. N.Y. Acad. Sci. 991, 189–198.
25. Taira, T., Saito, Y., Niki, T., Iguchi-Ariga, S. M., Takahashi, K. & Ariga, H. (2004) EMBO
Rep. 5, 213–218.
26. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W. & Vogelstein, B. (1998) Proc.
Natl. Acad. Sci. USA 95, 2509–2514.
27. Giovanni, A., Keramaris, E., Morris, E. J., Hou, S. T., O’Hare, M., Dyson, N., Robertson,
G. S., Slack, R. S. & Park, D. S. (2000) J. Biol. Chem. 275, 11553–11560.
28. Ghahremani, M. H., Keramaris, E., Shree, T., Xia, Z., Davis, R. J., Flavell, R., Slack, R. S.
& Park, D. S. (2002) J. Biol. Chem. 277, 35586–35596.
29. Cheung, N. S., Hickling, Y. M. & Beart, P. M. (1997) Neurosci. Lett. 233, 13–16.
30. Crocker, S. J., Smith, P. D., Jackson-Lewis, V., Lamba, W. R., Hayley, S. P., Grimm, E.,
Callaghan, S. M., Slack, R. S., Melloni, E., Przedborski, S., et al. (2003) J. Neurosci. 23,
31. Crocker, S. J., Lamba, W. R., Smith, P. D., Callaghan, S. M., Slack, R. S., Anisman, H. &
Park, D. S. (2001) Proc. Natl. Acad. Sci. USA 98, 13385–13390.
32. Smith, P. D., Crocker, S. J., Jackson-Lewis, V., Jordan-Sciutto, K. L., Hayley, S., Mount,
M. P., O’Hare, M. J., Callaghan, S., Slack, R. S., Przedborski, S., et al. (2003) Proc. Natl.
Acad. Sci. USA 100, 13650–13655.
33. Hayley, S., Crocker, S. J., Smith, P. D., Shree, T., Jackson-Lewis, V., Przedborski, S., Mount,
M., Slack, R., Anisman, H. & Park, D. S. (2004) J. Neurosci. 24, 2045–2053.
34. Matsuura, K., Kabuto, H., Makino, H. & Ogawa, N. (1997) J. Neurosci. Methods 73, 45–48.
35. Goldberg, M. S., Fleming, S. M., Palacino, J. J., Cepeda, C., Lam, H. A., Bhatnagar, A.,
Meloni, E. G., Wu, N., Ackerson, L. C., Klapstein, G. J., et al. (2003) J. Biol. Chem. 278,
36. Betarbet, R., Sherer, T. B., MacKenzie, G., Garcia-Osuna, M., Panov, A. V. & Greenamyre,
J. T. (2000) Nat. Neurosci. 3, 1301–1306.
37. Sherer, T. B., Betarbet, R., Testa, C. M., Seo, B. B., Richardson, J. R., Kim, J. H., Miller,
G. W., Yagi, T., Matsuno-Yagi, A. & Greenamyre, J. T. (2003) J. Neurosci. 23, 10756–10764.
38. Di Chiara, G. & Imperato, A. (1988) Proc. Natl. Acad. Sci. USA 85, 5274–5278.
39. Giros, B., Jaber, M., Jones, S. R., Wightman, R. M. & Caron, M. G. (1996) Nature 379,
40. Tatton, W. G., Kwan, M. M., Verrier, M. C., Seniuk, N. A. & Theriault, E. (1990) Brain Res.
41. Takahashi-Niki, K., Niki, T., Taira, T., Iguchi-Ariga, S. M. & Ariga, H. (2004) Biochem.
Biophys. Res. Commun. 320, 389–397.
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