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).

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 preliminarily obtained results that H₂O₂-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 H₂O₂-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).

In primary culture of rat ventral mesencephalon, treatment with 100 μM H₂O₂ 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 H₂O₂-treatment for 24 h (Figure 4B). H₂O₂-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).

In DJ-1-knockdown SH-SY5Y cells, the expression of endogenous DJ-1 protein was suppressed by 68% (Figure 4A), and massive H₂O₂-induced cell death occurred, compared to the results in normal SH-SY5Y cells (Figure 4B and C). Interestingly, UCP0045037 did not protect against H₂O₂-induced death in DJ-1-knockdown cells (Figure 4C).

In normal SH-SY5Y cells, incubation with 100 μM H₂O₂ for 1 h induced marked production of intracellular ROS (Figures 5A and B), while the production was only slightly induced by 50 μM H₂O₂ (Figure 6A). In DJ-1-knockdown cells, even 50 μM H₂O₂ 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 H₂O₂, 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 H₂O₂ (Figure 6B).

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 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.

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.

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.

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% CO₂/95% 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 H₂O₂-induced neurotoxicity, the cultures were also incubated simultaneously with the medium with 2% B-27 supplement minus antioxidant (Invitrogen) containing 100 μM H₂O₂ 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 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.

The human neuroblastoma cell line SH-SY5Y was cultured in DMEM supplemented with 10% FCS and kept at 37 °C in humidified 5% CO₂/95% 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 H₂O₂ (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.

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.).

To detect H₂O₂-induced ROS production, we used a redox-sensitive dye, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate acetyl ester (CM-H₂DCFDA; Molecular Probes) [22]. CM-H₂DCFDA 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-H₂DCFDA 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 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).

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 H₂O₂ 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).

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).