Int. J. Mol. Sci. 2009, 10, 4789-4804; doi:10.3390/ijms10114789

International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Oxidative Neurodegeneration Is Prevented by UCP0045037, an
Allosteric Modulator for the Reduced Form of DJ-1, a Wild-
Type of Familial Parkinson’s Disease-Linked PARK7
Koichiro Yamane
1
, Yoshihisa Kitamura
1,
*, Takashi Yanagida
1
, Kazuyuki Takata
1
, Daijiro
Yanagisawa
1
, Takashi Taniguchi
1
, Takahiro Taira
2
and Hiroyoshi Ariga
3,
*
1
Department of Neurobiology, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan;
E-Mails: ky04379@poppy.kyoto-phu.ac.jp (K.Y.); kaz@mb.kyoto-phu.ac.jp (K.T.);
brain@mb.kyoto-phu.ac.jp (T.T.)
2
Department of Molecular Cell Biology, Interdisciplinary Graduate School of Medicine and
Engineering, University of Yamanashi, Chuo 409-3898, Japan;
E-Mail: taira@yamanashi.ac.jp (T.T.)
3
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060-0812, Japan
* Authors to whom correspondence should be addressed; E-Mails: yo-kita@mb.kyoto-phu.ac.jp
(Y.K.); hiro@pharm.kokudai.ac.jp (H.A.); Tel.: +81-75-595-4706 (Y.K.); +81-11-706-3745 (H.A.);
Fax: +81-75-595-4796 (Y.K.); +81-11-706-4988 (H.A.).
Received: 17 September 2009; in revised form: 21 October 2009 / Accepted: 2 November 2009 /
Published: 5 November 2009

Abstract: Although a loss-of-function mutation has been identified in familial Parkinson’s
disease PARK7, the wild-type of DJ-1 is known to act as an oxidative stress sensor in
neuronal cells. Recently, we identified UCP0045037 as a compound that bound to the
reduced form of DJ-1 by in silico virtual screening. In this study, we determined the
neuroprotective effects of UCP0045037 against focal cerebral ischemia-induced
neurodegeneration in rats. Hydrogen peroxide-induced cell death was significantly
inhibited by UCP0045037 in both rat mesencephalic dopaminergic neurons and human
normal SH-SY5Y cells. In contrast, DJ-1-knockdown SH-SY5Y cells lost the protective
activity of UCP0045037. These results suggest that UCP0045037 interacts with
endogenous DJ-1 and produces a neuroprotective response.
OPEN ACCESS

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Keywords: DJ-1; allosteric modulator; neuroprotection; anti-oxidative response; in silico
virtual screening; in vivo rat brain; in vitro neuronal culture


1. Introduction

DJ-1 was first discovered as a novel oncogene product in collaboration with activated small GTP-
binding protein ras [1], and was later identified as a causative gene in the seventh type of familial
Parkinson’s disease, PARK7 [2]. Wild-type DJ-1 has many functions, and can act as oxidative stress
sensor, mitochondrial regulator, molecular chaperone, protease, regulator for gene transcription and
mRNA stability, and also stimulatory factor for spermatogenesis and fertilization [3–6]. X-ray
crystallographic and biologic analyses have shown that wild-type DJ-1 protein forms a homodimer. In
addition, these analyses have also shown that a substitution of leucine at amino acid position 166 by
proline (Leu166Pro-mutation) in DJ-1 protein, which was first found in a PARK7 patient, disrupts
dimer formation, resulting in a loss of function [7–11]. Furthermore, the cysteine residues in wild-type
DJ-1 protein are oxidized, and the isoelectric point (pI) of DJ-1 is shifted to being more acidic [12].
Thus, DJ-1 protein may play a key role in anti-oxidation and neuroprotection in neuronal cells
[9,13–15].
Wild-type DJ-1 in human and rat has three cysteine (Cys) residues at amino acid numbers 46, 53
and 106 (Cys46, Cys53 and Cys106, respectively) [12]. A Cys residue is oxidized from the reduced
form (-SH) to undergo sulphenation (-SOH), sulphination (-SO
2
H) and sulphonation (-SO
3
H), in order
of oxidative development. Among these three Cys residues, Cys106 is the most sensitive to oxidative
stress [12]. More recently, we used the X-ray crystal structure of DJ-1 at Cys106 with the reduced
form and the three-dimensional coordinate data of about 30,000 chemical compounds in the University
Compound Project (UCP) at the Foundation for Education of Science and Technology in Japan, and
performed in silico virtual screening to search for DJ-1-binding compounds (DJ-1 ligands). Among the
DJ-1 ligands identified in silico, UCP0045037 (Figure 1) had the highest binding constant (docking
score) toward the pocket of the reduced Cys106 region and exhibited neuroprotective effects in a
Parkinson’s disease model in rats, which were microinjected with 6-hydroxydopamine into the
substantia nigra [16]. However, it is not yet known whether UCP0045037 is effective in brain stroke.

Figure 1. Chemical structure of UCP0045037.

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Cerebral ischemia occurs as a result of a local reduction or arrest of the blood supply, and leads to
neuronal cell death in the ischemic region. Neurons and glial cells can be lethally damaged by several
events, such as peri-infarct depolarization, which occurs within hours after the onset of ischemia, and
the production of reactive oxygen species (ROS), which occurs immediately after
ischemia/reperfusion, followed by more delayed post-ischemic inflammation and apoptosis, and these
events are involved in the progression and expansion of brain injury [17,18]. This burst of ROS is
involved in direct cytotoxic effects, including protein and lipid peroxidation, oxidative DNA damage
and post-ischemic inflammatory injury through redox-mediated signaling pathways [19,20]. The
oxidation of proteins can have wide-ranging damaging effects, such as disruption of the active sites of
enzymes or alteration of the conformation of structural proteins. Thus, the components of ROS are
thought to contribute to neuronal loss after ischemia/reperfusion [19,20]. Therefore, the control of
ROS production is important for achieving neuroprotection against ischemia/reperfusion injury.
In the present study, to clarify the effects of UCP0045037 and DJ-1 on brain stroke and oxidative
insult, we examined the effects of UCP0045037 on focal ischemia/reperfusion-induced oxidative brain
damage in an in vivo neuronal model and on hydrogen peroxide (H
2
O
2
)-induced cell death in rat
ventral mesencephalic neurons; or in normal and DJ-1-knockdown SH-SY5Y cells in an in vitro
cultured neuronal model.

2. Results

2.1. Effect of DJ-1 Ligand in the in Vivo Rat Brain

We previously found that neuronal death induced by occlusion of the middle cerebral artery (MCA)
was significantly inhibited by the intrastriatal microinjection of recombinant protein of wild-type
human DJ-1 [15]. Therefore, to clarify the effect of the DJ-1 ligand UCP0045037, which can bind to
the pocket region at the reduced Cys106 of DJ-1 protein [16], in the in vivo ischemic brain, we
performed an intrastriatal microinjection of this DJ-1 ligand and then subjected to the animals to 90
min of MCA occlusion (MCAO) and reperfusion (Figure 2).
Since UCP0045037 was hard to be solved by water, we prepared UCP0045037 solution at 1 mM
(containing 1% dimethyl sulfoxide, DMSO) in sterilized physiological saline. At 24 h after MCAO, a
marked decrease in the area and volume stained with 2,3,5-triphenyltetrazolium chloride (TTC)
occurred in the ipsilateral cerebral cortex and striatum in rats, which were microinjected with 4 µL of
sterilized physiological saline containing 1% DMSO (vehicle). In contrast, the area of TTC-staining
lost was smaller with the intrastriatal microinjection of UCP0045037 (4 nmol/4 µL containing 1%
DMSO) (Figure 2A). In the quantitative analysis, each infarct area was smaller and the total infarct
volume was significantly reduced by the administration of UCP0045037, compared to that in vehicle-
injected rats (Figures 2B and C).

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Figure 2. DJ-1 ligand UCP0045037 reduces infarct size after focal cerebral ischemia and
reperfusion. (A) Representative photographs showing coronal brain sections at +5, +3, +1,
–1, –3, –5, and –7 mm anterior-posterior from the bregma with TTC staining at 1 day after
MCAO in sham-operated rats (n = 5) and MCAO-ischemic rats injected with sterilized
physiological saline in the presence of the vehicle (4 µL, 1% DMSO, n = 5) or
UCP0045037 (4 nmol/4 µL containing 1% DMSO, n = 5), at 30 min before MCAO
(90 min). (B, C) Quantitative analysis of infarct area and volume, respectively. Data are the
mean±SEM. Significance (Bonferroni/Dunn post hoc comparisons after ANOVA):
***P < 0.001 vs. sham-operated rats. †P < 0.05, †††P < 0.001 vs. vehicle-injected rats.
Scale bar: 1 mm (Sham in A).

2.2. Effect of DJ-1 Ligand in the in Vitro Neuronal Cultures

We have previously found that MCAO-induced focal ischemia retrogradely causes loss and damage
of dopaminergic neurons in the rat substantia nigra, and results in behavioral and motor dysfunction
[21]. It is well known that nigrostriatal dopaminergic neurons are specifically lost in the brains of
patients with Parkinson’s disease. While, SH-SY5Y cells are frequently used as the human
dopaminergic neuronal model [22]. In the present study, therefore, we focus effect of UCP0045037 on
dopaminergic neurons, using in vitro rat and human dopaminergic neuronal models. In brief, we
performed primary culture of dopaminergic neurons, which were prepared from the ventral two-thirds
of the mesencephalon of rat embryos on the 16th days of gestation, as described previously [23]. We

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preliminarily obtained results that H
2
O
2
-induced cell death in normal SH-SY5Y cells was markedly
inhibited by 10 µM UCP0045037, while moderately by 1 µM, but not by 0.1 µM (data not shown).
Based on these observations, we examined effect of UCP0045037 at 10 µM on H
2
O
2
-induced cell
death (and ROS production) in the rat primary-cultured neurons (and human SH-SY5Y cells) in
Figure(s) 3 (and 4-6, respectively).

Figure 3. Protective effect of UCP0045037 on hydrogen peroxide-induced dopaminergic
neuronal death. Rat ventral mesencephalic cultured neurons, prepared from day-16
embryos, were treated with 100 µM H
2
O
2
in the presence of the vehicle (0.01% DMSO) or
UCP0045037 at 10 µM (containing 0.01% DMSO) for 24 h. Subsequently, treated neurons
were fixed and immunostained by anti-TH antibody and then the number of TH-
immunopositive dopaminergic neurons was counted in the cultured area (1.9 mm
2
). (A, B,
C) Immunoreactivity of dopaminergic neurons treated with vehicle, H
2
O
2
alone, and H
2
O
2

and UCP0045037, respectively. Scale bar: 100 µm. (D) Semi-quantitative analysis of
dopaminergic neurons. Data are the mean±SEM of four determinations. Significance
(Bonferroni/Dunn post hoc comparisons after ANOVA): ***p < 0.001 vs. vehicle-
treatment. †††p < 0.001 vs. H
2
O
2
-treatment.

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4794
In primary culture of rat ventral mesencephalon, treatment with 100 µM H
2
O
2
for 24 h significantly
caused the death of dopaminergic neurons, which were immunostained by anti-tyrosine hydroxylase
(TH) antibody (Figures 3A, B and D). Similarly, wild-type (normal) human SH-SY5Y cells also died
upon H
2
O
2
-treatment for 24 h (Figure 4B). H
2
O
2
-induced death of either rat dopaminergic neurons or
normal human SH-SY5Y cells was significantly inhibited by simultaneous treatment with 10 µM
UCP0045037 (Figures 3C, D and 4B).

Figure 4. Effects of UCP0045037 on hydrogen peroxide-induced death of human SH-
SY5Y cells. (A) Expression of endogenous DJ-1 protein. The intensity of the expression
level in normal cells was set to 1.00. (B, C) Normal and DJ-1-knockdown SH-SY5Y cells
(B and C, respectively) were treated with 100 µM H
2
O
2
in the presence of the vehicle
(0.01% DMSO) or UCP0045037 at 10 µM (containing 0.01% DMSO) for 24 h.
Subsequently, cell viability was measured by a color metrical assay using 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Data are the mean±SEM of three
determinations. Significance (Bonferroni/Dunn post hoc comparisons after ANOVA):
***p < 0.001 vs. vehicle-treatment. †p < 0.05 vs. H
2
O
2
-treatment in normal SH-SY5Y
cells. ##p < 0.01 vs. H
2
O
2
-treatment in the presence of UCP0045037 in normal SH-SY5Y
cells.











In DJ-1-knockdown SH-SY5Y cells, the expression of endogenous DJ-1 protein was suppressed by
68% (Figure 4A), and massive H
2
O
2
-induced cell death occurred, compared to the results in normal
SH-SY5Y cells (Figure 4B and C). Interestingly, UCP0045037 did not protect against H
2
O
2
-induced
death in DJ-1-knockdown cells (Figure 4C).
In normal SH-SY5Y cells, incubation with 100 µM H
2
O
2
for 1 h induced marked production of
intracellular ROS (Figures 5A and B), while the production was only slightly induced by 50 µM H
2
O
2

(Figure 6A). In DJ-1-knockdown cells, even 50 µM H
2
O
2
induced significant fluorescence intensity
after 1 h (Figures 6A and B). On the other hand, treatment with 10 µM UCP0045037 significantly
inhibited production of ROS, which had been induced by 100 µM H
2
O
2
, in normal SH-SY5Y cells
(Figure 5B). Unfortunately, in DJ-1-knockdown cells, this DJ-1 ligand did not have an inhibitory effect
on ROS production induced by 50 µM H
2
O
2
(Figure 6B).

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Figure 5. Effects of UCP0045037 on hydrogen peroxide-induced production of ROS in
human SH-SY5Y cells. Normal SH-SY5Y cells were treated with 100 µM H
2
O
2
in the
presence of the vehicle (0.01% DMSO) or UCP0045037 at 10 µM (containing 0.01%
DMSO) for 1 h. Subsequently, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein
diacetate acetyl ester (CM-H
2
DCFDA) was added. Fluorescence of oxidized 2’,7’-
dichlorofluorescein (DCF, green) was visualized, and then the intensity was measured as
ROS production. (A) ROS production-dependent DCF fluorescence in normal SH-SY5Y
cells. Nuclei were stained with Hoechst33258 (blue) and cellular images (cell shapes) were
obtained by the difference interference contrast (DIC). (B) The relative fluorescence
intensity was quantified by computerized image analysis. Data are the mean±SEM of five
determinations, based on the intensity in normal cells treated with H
2
O
2
alone as 100%.
Significance (Bonferroni/Dunn post hoc comparisons after ANOVA): **p < 0.01,
***p < 0.001 vs. control. ††p < 0.01 vs. H
2
O
2
-treatment.

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4796
Figure 6. Loss of the inhibitory effect of UCP0045037 on hydrogen peroxide-induced
production of ROS in DJ-1-knockdown cells. DJ-1-knockdown and normal SH-SY5Y cells
were treated with 50 µM H
2
O
2
in the presence of the vehicle (0.01% DMSO) or
UCP0045037 at 10 µM (containing 0.01% DMSO) for 1 h, and then CM-H
2
DCFDA was
added. Subsequently, ROS production-dependent DCF fluorescence was analyzed. (A)
ROS production-dependent fluorescence in normal and DJ-1-knockdown SH-SY5Y cells,
which were treated with 50 µM H
2
O
2
. Nuclei were stained with Hoechst33258 (blue) and
cell shapes were obtained by DIC. (B) The relative fluorescence intensity was quantified
by computerized image analysis. Data are the mean±SEM of three determinations, based
on the intensity in DJ-1-knockdown SH-SY5Ycells treated with 50 µM H
2
O
2
alone as
100%. Significance (Bonferroni/Dunn post hoc comparisons after ANOVA): ***p < 0.001
vs. control. †††p < 0.001 vs. H
2
O
2
-treatment in DJ-1-knockdown cells.















3. Discussion

Although oxygen is necessary for life, it paradoxically generates ROS, which are highly toxic to
cells, as a by-product of its metabolism. In addition, ROS are massively produced in the brain after

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cerebral ischemia and reperfusion. Oxidative stress can be defined as an imbalance between ROS
generation and the anti-oxidant capacity of a cell. Recent studies, together with our experiments,
indicate that DJ-1 may sense oxidative stress. We previously found that treatment with recombinant
human DJ-1 protein protected against cell death of nigrostriatal dopaminergic neurons in
6-hydroxydopamine-treated Parkinson’s disease model rats and MCAO-ischemic rats [9,15].
Protein oxidation indicates the trapping of oxygen atoms (Os) to amino acid residues, i.e.,
methionine (Met) and Cys are oxidized to Met-SO (1O) and Cys-SO
3
H (3Os), respectively, and rat and
human DJ-1 proteins have four Met and three Cys residues [24]. Therefore, if these amino acid
residues are fully oxidized, one molecule of DJ-1 protein has a potential total of an additional 13Os. In
addition, recent studies have indicated that a reduced form and/or partial oxidation at Cys106 induce
DJ-1 activation, which may exert an antioxidative response, while massive oxidation may cause a loss-
of-function of DJ-1 [13,24]. Thus, the reduced form (-SH) and/or moderate oxidation (-SOH and/or -
SO
2
H) at Cys106 may produce active forms of DJ-1 (as a sensor of oxidative stress), while higher
peroxidation (-SO
3
H) causes loss-of-function and produces the inactive form.
By in silico virtual screening, we recently identified UCP0045037 as a DJ-1-binding ligand, which
can bind to the region around the reduced Cys106 form. In addition, direct binding between
UCP0045037 and a recombinant wild-type DJ-1 protein (but not a mutated DJ-1 which has a
substitution of Cys106 by serine, bovine serum albumin, or glutathione-S-transferase) was detected by
a biosensor chip of a quartz crystal microbalance system [16], which suggests that the binding of
UCP0045037 is relatively specific to the wild-type DJ-1 protein and its binding site may be the
Cys106 region. Furthermore, matrix-assisted laser desorption/ionization (MALDI)-time of flight
(TOF)/TOF-mass spectrometry (MS) analysis indicated that the H
2
O
2
-induced formation of
hyperoxidative C106-SO
3
H was inhibited by UCP0045037 [16]. On the other hand, DJ-1 is relatively
rich in neurons and glial cells, except for nigral dopaminergic neurons [14]. In addition to the presence
of DJ-1 in the cytoplasm, mitochondria and nucleus, recent studies have reported that DJ-1 protein was
detected in extracellular spaces, such as plasma and cerebrospinal fluids [25–28], suggesting that DJ-1
protein may function in both intracellular and extracellular spaces. Therefore, we consider that the
amount of endogenous DJ-1protein is enough for the function of UCP0045037 in rat brain in vivo and
in normal SH-SY5Y cells in vitro.
In the present study, we revealed that the intrastriatal microinjection of UCP0045037 inhibited
neurodegeneration induced by 90 min of MCAO and reperfusion in rats. In rat mesencephalic primary
culture, UCP0045037 protected against dopaminergic neuronal death induced by 100 µM H
2
O
2
. In
normal SH-SY5Y cells, treatment with UCP0045037 reduced production of ROS and cell death
induced by 100 µM H
2
O
2
. In DJ-1-knockdown cells, H
2
O
2
at an even lower concentration (50 µM)
significantly induced ROS production and cell death. However, DJ-1 knockdown caused a loss of the
inhibitory effects of UCP0045037 toward ROS production and cell death. In DJ-1-knockdown SH-
SY5Y cells in this study, endogenous DJ-1 protein partially remained (about 32%), but UCP0045037
did not affect in the knockdown cells. We presume that since residual 32% of DJ-1 protein might be
almost completely SO
3
H-peroxidized by H
2
O
2
, simultaneous treatment with UCP0045037 could not
inhibit SO
3
H-peroxidation of Cys106 in the knockdown cells. In normal SH-SY5Y cells, although
DJ-1 protein was partially SO
3
H-peroxidized, the reduced and/or SOH/SO
2
H-oxidation forms of
Cys106 might remain under treatment with UCP0045037. Therefore, UCP0045037 treatment may

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induce anti-oxidative and neuroprotective responses in normal cells. Based on these observations, we
consider that endogenous DJ-1 protein is necessary for the antioxidant and neuroprotective effects
induced by UCP0045037. In brief, UCP0045037 binds to DJ-1 protein as an allosteric modulator.
Furthermore, since UCP0045037 binds to the reduced Cys106 region in wild-type DJ-1 protein, it is
possible that UCP0045037 stabilizes the reduced DJ-1 form or mimics SOH/SO
2
H-oxidized Cys106,
and inhibits peroxidation to inactive DJ-1 with SO
3
H-oxidized Cys106.
Recent studies have suggested that DJ-1 induces indirect antioxidant episodes, which are mediated
through the stabilizing nuclear factor erythroid 2-related factor (Nrf2) by preventing association with
the Kelch-like erythroid cell-derived protein with cap‘n’collar homology (ECH)-associated protein-1
(Keap1), and this activates antioxidant transcriptional responses, such as the induction and enhanced
expression of NAD(P)H quinine oxidoreductase-1 (NQO1), heme oxigenase-1, superoxide
dismutase-1, thioredoxin, and so on [29].
On the other hand, DJ-1 inhibits several apoptotic pathways, such as the pyrimidine tract-binding
protein-associated splicing factor (PSF)/54-kDa nuclear RNA-binding protein (p54nrb) pathway [30]
and/or apoptosis signal regulating kinase 1 (ASK1)/homeodomain-interacting protein kinase
(HIPK1)/death-domain-associated protein (Daxx) pathway [31,32]. Thus, DJ-1 protein may induce: (i)
an immediate antioxidant response by the direct trapping of oxygen atoms into amino acids; (ii) early
indirect antioxidant responses, probably by the Nrf2-mediated induction of antioxidant
enzymes/molecules, and (iii) delayed anti-apoptotic responses. Based on these observations, we
consider that UCP0045037 may maintain and enhance DJ-1-induced anti-oxidative and anti-apoptotic
activation and these responses may synergistically act to prevent both ROS production and neuronal
cell death. Therefore, the interaction of 10 µM UCP0045037 with endogenous DJ-1 could inhibit 100
µM H
2
O
2
-induced ROS production and cell death in the in vitro culture model. These studies raise the
possibility that UCP0045037 is a new type of neuroprotective drug for the treatment of brain ischemia.

4. Experimental Section

4.1. In Vivo Model of Rat Focal Cerebral Ischemia

Wistar rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The animals were
acclimated to and maintained at 23 °C under a 12-h light/dark cycle (lights on 08:00–20:00 hours).
Rats were housed in standard laboratory cages and had free access to food and water throughout the
study period. All animal experiments were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the
Committee for Animal Research at Kyoto Pharmaceutical University. Male Wistar rats weighing
260–300 g were used in the present study. Focal cerebral ischemia was induced by the intraluminal
introduction of a nylon thread as described previously [15,33]. Briefly, animals were anesthetized with
4% halothane (Takeda Pharmaceutical, Osaka, Japan), and maintained on 1.5% halothane using a
facemask. After a midline neck incision was made, 20 mm of 4-0 nylon thread with its tip rounded by
heating and coated with silicone (Xantopren M; Heraeus Kulzer, Hanau, Germany) was inserted into
the left internal carotid artery (ICA) as far as the proximal end using a globular stopper. The origin of
the middle cerebral artery (MCA) was then occluded by a silicone-coated embolus. Anesthesia was

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4799
discontinued, and the development of right hemiparesis with upper limb dominance was used as the
criterion for ischemic insult. After 90 min of MCA occlusion (MCAO), the embolus was withdrawn to
allow reperfusion of the ischemic region via the anterior and posterior communicating arteries. Body
temperature was maintained at 37–37.5 °C with a heating pad and lamp during surgery. In the sham
operation, a midline neck incision was made to expose the arteries, but the nylon thread was not
inserted into the carotid artery.

4.2. Intrastriatal Drug Administration to Ischemic Rats

Male Wistar rats (SLC, Shizuoka), weighing approximately 300 g, were used. Under deep
anesthesia (sodium pentobarbital, 50 mg/kg, i.p.), rats received a microinjection of UCP0045037
(4 nmol/4 µL) in the left striatum (coordinates: 1 mm anterior, 4 mm left lateral, and 5 mm ventral
from the bregma). Sterilized physiological saline containing 1% dimethyl sulfoxide (DMSO) was used
as the vehicle control in a final volume of 4 µL. After 30 min, left MCAO for 90 min and reperfusion
were performed.

4.3. Measurement of Infarct Volume in Rat Ischemic Brain

At 24 h after MCAO, brains were removed and cut into 2-mm-thick coronal sections. These
sections were immersed in 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Wako Pure
Chemical Industries, Osaka, Japan) in saline at 37 °C for 20 min, and then fixed in 4%
paraformaldehyde in 100 mM phosphate buffer (PB) at 4 °C, and infarct areas and volumes were
quantified.

4.4. Primary Neuronal Culture of Rat Ventral Mesencephalon

Cultures of the rat mesencephalon were established according to methods described previously [23].
The ventral two-thirds of the mesencephalon were dissected from rat embryos on the 16th days of
gestation. The dissected regions included dopaminergic neurons from the substantia nigra and the
ventral tegmental area but not noradrenergic neurons from the locus ceruleus. Neurons were dissected
mechanically and plated out onto 0.1% polyethyleneimine-coated 24-well plates at a density of
approximately 2,500,000 cells/well. The culture medium consisted of Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 50 μg/mL penicillin and
100 μg/mL streptomycin and kept at 37 °C for two days in humidified 5% CO
2
/95% air, and DMEM
containing 2% B-27 supplement (Invitrogen, Carlsbad, CA, USA) and 2 mg/mL aphidicolin (Sigma, St.
Louis, MO, USA) without FCS from the third day onwards. To examine the effect of UCP0045037 on
H
2
O
2
-induced neurotoxicity, the cultures were also incubated simultaneously with the medium with
2% B-27 supplement minus antioxidant (Invitrogen) containing 100 μM H
2
O
2
in the presence of
vehicle (0.01% DMSO) or 10 µM UCP0045037 (containing 0.01% DMSO) for 24 h on the 7th day of
culture, and then fixed. Control experiments were sham operations, similar to the treatment but with
the medium containing no drug. After fixation, cultured cells were incubated with anti-tyrosine
hydroxylase (TH) antibody (diluted at 1:3,000; Chemicon International, Temecula, CA, USA) for

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4800
approximately 24 h at 25 °C. The cultured cells were then incubated with avidin peroxidase (1:4,000;
ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) for 1 h at 25 °C. The cultured cells were
washed several times with 100 mM phosphate-buffered saline containing 0.3% Triton X-100 (PBS-T)
between each incubation, and labeling was revealed by 3,3’-diaminobenzidine with nickel
enhancement, which yielded a dark blue color. TH-immunopositive neurons were then counted.

4.5. In Vitro Cell Culture and Establishment of DJ-1-Knockdown Cells

The human neuroblastoma cell line SH-SY5Y was cultured in DMEM supplemented with 10% FCS
and kept at 37 °C in humidified 5% CO
2
/95% air. We established a DJ-1-knockdown SH-SY5Y cell
line by a method that was essentially similar to that in mouse Flp-In3T3 cells [34]. In brief, the
nucleotide sequence of the upper strand of the oligonucleotide used to construct an siRNA vector that
targets the human DJ-1 gene is 5’-GGA TCC CGT CAA GGC TGG CAT CAG GAC AAT TGA TAT
CCG TTG TCC TGA TGC CAG CCT TGA TTT TTT CCA AAA GCT T-3’. After oligonucleotides
corresponding to the upper and lower strands of DNA were annealed, they were inserted into BamHI-
HindIII sites of pRNA-U6.1/Neo. These plasmids were transfected into human SH-SY5Y cells by the
calcium phosphate precipitation method, and the cells were cultured in RPMI-1640 medium in the
presence of 400 μg/mL G418 for 14 days. Cells that were resistant to the drug were selected, and the
intrinsic expression of DJ-1 was checked by western blotting with an anti-human DJ-1 antibody. In
addition, normal and DJ-1-knockdown SH-SY5Y cells were treated with various concentrations of
H
2
O
2
(at 50 µM or 100 µM) in the presence of vehicle (0.01% DMSO) or 10 µM UCP0045037
(containing 0.01% DMSO) for 24 h.

4.6. Western Blotting Analysis

Cell lysates were diluted with Laemmli’s sample buffer and subjected to sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (15% polyacrylamide gels), and immunoblotting was then carried
out using antibodies against human DJ-1 (1:5,000) and β-actin (10,000). For a semi-quantitative
analysis, the bands of these proteins on radiographic films were scanned with a CCD color scanner
(DuoScan; AGFA, Leverkusen, Germany), and then analyzed. Densitometric analysis was performed
using the public domain program NIH Image 1.56 (written by Wayne Rasband at the US National
Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh. nih.gov.).

4.7. Assay of ROS Production

To detect H
2
O
2
-induced ROS production, we used a redox-sensitive dye, 5-(and-6)-chloromethyl-
2’,7’-dichlorodihydro-fluorescein diacetate acetyl ester (CM-H
2
DCFDA; Molecular Probes) [22].
CM-H
2
DCFDA is readily taken up by cells. When it reacts with ROS, it is converted to oxidized
2’,7’-dichlorofluorescein (DCF), and subsequently illuminates at an excitation wavelength of 488 nm.
After SH-SY5Y cells were prepared in uncoated glass-bottomed microwells (inner diameter, 18 mm),
CM-H
2
DCFDA was added to the cell culture to a final concentration of 2 µmol/L for 10 min at 37 °C.
After two rinses with serum-free medium, the fluorescence intensity of DCF was scanned under a

Int. J. Mol. Sci. 2009, 10


4801
confocal microscope (LSM410, Carl Zeiss, Jena, Germany). Since illumination at an excitation
wavelength of 488 nm causes increased fluorescence due to oxidation of this dye [22], each field was
exposed to light for exactly the same amount of time. Fluorescence intensity was quantified by
computerized image analysis (WinRoof, Mitani. Fukui, Japan).

4.8. Measurement of Cell Viability

To evaluate cell viability, we performed a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT; Dojindo Laboratories) assay as an index of surviving cells. In living cells, MTT is
converted to formazan, which has a specific absorption maximum. SH-SY5Y cells, in which
endogenous DJ-1 expression was normal or knocked-down by siRNA vector, were seeded at
30,000 cells/well in a 96-microwell plate, and treated with H
2
O
2
24 h after plating. At 24 h after
treatment, the culture medium was changed to a medium containing 5 mg/mL MTT, and the cells were
incubated for an additional 4 h. They were then mixed thoroughly with an equal volume of
isopropanol/0.04 M HCl, and sonicated to completely dissolve formazan. Absorbance was then
measured at 570 nm with a microplate reader (BioRad Laboratories, Hercules, CA, USA).

4.9. Statistical Analysis

Results are presented as the mean ± standard error of the mean (SEM). The significance of
differences was determined by an analysis of variance (ANOVA). Further post hoc comparisons were
performed using Bonferroni/Dunn tests (Stat View; Abacus Concepts, Berkeley, CA, USA).

5. Conclusions

This is the first report to show that UCP0045037 has a neuroprotective effect in an in vivo animal
model of brain ischemia. UCP0045037 may bind to the reduced C106 region in endogenous DJ-1
protein and then maintain a DJ-1-induced neuroprotective response. The present results suggest that
allosteric modulators for DJ-1 (DJ-1 ligands), such as UCP0045037, may be useful for neuroprotective
treatment in cytoprotective therapy for various oxidative stress-mediated disorders.

Acknowledgements

This study was supported in part by Frontier Research Programs from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), and by the Program for Promotion of Fundamental
Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) in Japan.

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