N-[4-(8-methyl(4-hydroimidazo[1,2-a]pyridin-2-yl))phenyl](3,4,5-trimethoxyphenyl)carboxamide, which is DJ-1-binding compound-23 (comp-23), was synthesized and obtained by Enamine Ltd. (Kiev, Ukraine). 6-Hydroxydopamine (6-OHDA) and DCFH-DA were purchased from Sigma (St. Louis, MO, USA) and from Invitrogen (Carlsbad, CA, USA), respectively. Mouse anti-tyrosine hydroxylase (TH), chicken anti-TH and anti-NeuN antibodies were purchased from Sigma, Chemicon (Temecula, CA, USA) and Chemicon, respectively. The ABC Elite kit from Vector Laboratories (Burlingame, CA, USA) was used. Methamphetamine was obtained from Dainippon Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan).
Human SH-SY5Y and its DJ-1-knockdown cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum. Establishment of DJ-1-knockdown SH-SY5Y cells was described previously .
Screening of DJ-1-binding compounds
Information on the X-ray crystal structures of reduced DJ-1 and oxidized DJ-1 at C106 as an SO2H form was obtained from a web site (http://www.rcsb.org/pdb/). To obtain the structure of DJ-1 containing H2O, the X-ray crystal structure of DJ-1 was modified using BioMedCAChe software (Fijitsu, Tokyo, Japan). Compounds were screened by targeting C106 of this structure on FastDock software (Fijitsu) in BioServer hardware (Fujitsu) according to the manufacturer's protocol. Briefly, the BioServer hardware used is PC clusters with 40 core of CPU of Xeon5355 (Fujitsu), OS of Red Hat 3.4.5-2 (Linux version 2.6.9-34) and 1.0 TB Hard Disk. The other conditions were exactly the same as those described previously .
Cell viability assay
Cells were cultured in a 96-well plate and treated with various amounts of hydrogen peroxide or 6-OHDA. Cell viability was then measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a cell counting kit -8 (DOJINDO, Osaka, Japan).
Binding of compound-23 to DJ-1 by a quartz crystal microbalance
Fixation of compounds on a sensor chip of QCM (Affinix Q, Initium, Tokyo, Japan) was carried out as follows. The sensor chip was washed with a solution containing H2O2 and sulfonic acid (molar ratio = 1:3), and then it was incubated with 4 μL of 1 μM compound dissolved in chloroform until the solution had evaporated. To the sensor chips fixed with compounds in Affinix Q, 8 μL of 1 μg/μL DJ-1 was applied, and their frequency was measured according to the manufacturer's protocol.
Primary neuronal culture of the ventral mesencephalon
Cultures of the rat mesencephalon were established according to methods described previously . The ventral two-thirds region of the mesencephalon was dissected from rat embryos on the 17-19th 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 dissociated mechanically and plated out onto 0.1% polyethyleneimine-coated 24-well plates at a density of 2.5 × 106 cells/well. The culture medium consisted of DMEM containing 10% fetal calf serum for 2 days and DMEM containing 2% B-27 supplement (Invitrogen) and 2 μg/mL aphidicolin (Sigma) without fetal calf serum from the third day onwards. The animals were treated in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals. After fixation, cultured cells were incubated with chicken anti-TH (diluted at 1:200) and anti-NeuN (1:200) antibodies for 24 hours at 25°C. The cells were also stained with 4',6-diamidino-2-phenylindole (DAPI). The cells were then reacted with a rhodamine-conjugated anti-rabbit IgG or fluorescein isothiocyanate-conjugated anti-mouse IgG and observed under an All-in-on microscope (Biorevo BZ-9000, Keyence).
To examine the effects of DJ-1-binding compounds on oxidative stress-induced cell death, the cells were cultured in the presence or absence of 1 μM of each compounds for 20 hours and then treated with 200 μM H2O2 for 3 hours. Cell viabilities were then examined by an MTT assay.
Detection of production of ROS
8 × 105 SH-SY5Y cells in a 96-well plate were pretreated with 1 μM of comp-23 for 20 hours and then treated with 40 μM 6-OHDA for 10 min after the addition of 10 μM DCFA-DA (Invitrogen) for 15 min. The amounts of ROS in cells were measured using a fluorescence spectrophotometer at extension of 485 nm and emission of 530 nm.
SH-SY5Y cells were incubated with 1 μM compound-23 or compound-B for 24 hours and then treated with various amounts of H2O2 for 10 min. Proteins extracted from the cells were separated in the pH 5-8 range of isoelectric focusing phoresis gel, transferred to nitrocellulose membranes, and blotted with an anti-DJ-1 polyclonal antibody as described previously .
SH-SY5Y cells in 6-well plates were incubated with 1 μM compound-23 or compound-B for 20 hours and then treated with various amounts of H2O2 for 3 hours. Cells were then treated with 0.5 mM DSS or DMSO for 30 min, and proteins extracted from cells were analyzed by Western blotting with an anti-DJ-1 antibody.
The hydroxyl radical (.OH) was monitored by ESR spectrometry with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Labotec Ltd., Tokyo, Japan), a spin trapper. In a final volume of 200 μL of 100 mM phosphate buffer (PB), comp-23 (1-100 μM) or thiourea (500 mM) was added to the reaction mixture containing diethylene-triamine pentaacetic acid (25 μM), FeSO4 (25 mM), H2O2 (100 μM), and DMPO (112.5 mM). These drugs and reagents were solubilized in Milli Q water. The reaction mixture was transferred to a flat quartz cuvette and placed in the cavity of an X-band JEOL RFR-30bRadical Analyzer system (JEOL Ltd., Tokyo, Japan). The .OH, which was generated by Fenton's reaction between Fe2+ and H2O2, was trapped by DMPO, and a stable adduct DMPO-OH was measured exactly 1 min after the addition of DMPO. The Mn2+-derived split signal was used as the internal standard. Typical instrumental stettings were as follows: incident-microwave of 4 mV, modulation-amplitude of 0.1 mT, time-constant of 0.10 s, and sweep rate of 5 mT/min.
Male Wistar rats (SLC, Shizuoka, Japan) weighing approximately 250 g were used. Rats were acclimated to and maintained at 23°C under a 12-hour light and dark cycle (light on 08:00-20:00 hours). 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. For stereotaxic microinjection, rats were anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) and immobilized in a Kopf stereotaxic frame. Subsequently, rats were simultaneously injected with 6-OHDA (32 nmol/4 μL) in the presence or absence of comp-23 (4 nmol/4 μL), in a final volume of 4 μL of physiological saline containing 0.02% ascorbic acid (as a 6-OHDA stabilizer) and 1% dimethyl sulfoxide (DMSO, as a solvent for comp-23). As a vehicle control, sterilized physiological saline containing 0.02% ascorbic acid and 1% DMSO was injected without 6-OHDA. The intranigral injection coordinates 4.8 mm anterior-posterior, 1.8 mm left lateral, and 7.8 mm ventral from the bregma were taken from a rat brain atlas. Injection was performed by a motor-driven 10-μl Hamilton syringe using a 26-gauge needle. The infusion rate was 1 μL/min, and the injection needle was kept in place for a further 5 min after injection. At the end of the experiments, all rats were sacrificed for immunohistochemical assessments.
Assay of rotational behavior
We used methamphetamine as a dopamine releaser . Drug-induced rotational asymmetry was assessed in rotometer bowls as described previously [20, 23, 39]. Briefly, the number of full body turn rotations in the ipsilateral direction was counted after the administration of methamphetamine (2.5 mg/kg, i.p., for 70 min).
Tissue preparation and immunohistochemistry
After assay of rotational behaviour, treated rats were perfused through the aorta with 150 mL of 10 mM PBS, followed by 300 mL of a cold fixative consisting of 4% paraformaldehyde in 100 mM phosphate buffer (PB) under deep anesthesia with pentobarbital (100 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with paraformaldehyde in 100 mM PB and then transferred to 15% sucrose solution in 100 mM PB containing 0.1% sodium azide at 4°C. The brain was cut into 20-μm-thick slices using a cryostat and collected in 100 mM PBS containing 0.3% Triton X-100 (PBS-T). Brain slices were incubated with a mouse anti-TH antibody (1:10,000, dilution) for 3 days at 4°C. After several washes, sections were incubated with biotinylated anti-mouse IgG antibody (1:2,000), as appropriate, for 2 hours at room temperature. The sections were then incubated with avidin peroxidase (1:4,000; ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature. All of the sections were washed several times with PBS-T between each incubation, and labeling was then revealed by 3,3'-diaminobenzidine (DAB) with nickel ammonium, which yielded a dark blue colour [20, 23].
Measurement of immunoreactive neurons and areas
The number of TH-immunopositive neurons in the substantia nigra and the optical density of TH-immunoreactive areas in the striatum were measured by a computerized image analysis system (WinRoof; Mitani, Tokyo, Japan) with a CCD camera (ProgRes 3008, Carl Zeiss, Jena, Germany) as described previously [20, 23]. The number of TH-immunopositive neurons in the substantia nigra was counted bilaterally on six adjacent sections between 4.6 and 4.9 mm posterior from the bregma. For each animal, neuronal survival in the substantia nigra was then expressed as the percentage of TH-immunopositive neurons on the lesioned side, with respect to the contralateral, intact side; this approach was chosen to avoid methodological biases because of interindividual differences and is widely used to assess the extent of a 6-OHDA-induced lesion in the substantia nigra [40–42].
For the analysis of striatal TH-immunoreactive intensity, the striatum was divided into anatomo-functional quadrants encompassing the dorsal (D), lateral (L), ventral (V), and medial (M) regions [41, 43] and the optical density was measured within a fixed box (0.5 × 0.5 mm) positioned approximately in the middle of these quadrantal parts. Immunoreactive intensity was expressed as percentage of the intensity recorded from the same area on the contralateral side [40, 43, 44]. Subsequently, the average of relative intensities in each quadrant was estimated from striatal slices (at 0.60 mm anterior from the bregma) and then statistical values were evaluated from treated rats.
In vivo model of rat focal cerebral ischemia
Male Wistar rats weighing 260-300 g were used. Focal cerebral ischemia was induced by the intraluminal introduction of a nylon thread as described previously . 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 had been 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 or 120 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.
Intrastriatal drug administration to ischemic rats
Ninety-min-MCAO-ischemic rats (SLC, Shizuoka) were used. Under deep anesthesia (sodium pentobarbital, 50 mg/kg, i.p.), rats received a microinjection of comp-23 (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% 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.
Intraperitoneal drug administration to ischemic rats
One hundred twenty-min-MCAO-ischemic rats were used. Animals were intraperitoneally administered with comp-23 (0.1, 1 and 10 mg/kg), before 10 min and after 2 hours of the reperfusion from MCAO. Sterilized physiological saline containing 1% DMSO was used as a vehicle control.
Measurement of infarct volume in rat ischemic brain
At 24 hours 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.
Rotenone-treated PD model mice and rota-rod test
Rotenone (Sigma, St. Louis, MO, USA) was administered orally once daily at a dose of 30 mg/kg for 56 days, as described previously [30, 31]. Rotenone was suspended in 0.5% carboxymethyl cellulose sodium salt (CMC, Nacalai Tesque, Kyoto, Japan) and administered orally once daily at a volume of 5 mL/kg body weight. 0.5% CMC was administered orally as vehicle to control mice.
Behaviour of each mouse was assessed by the rota-rod test, as also described previously [30, 31]. The rota-rod treadmill (accelerating model 7750, Ugo Basile, Varese, Italy) consists of a plastic rod, 6 cm in diameter and 36 cm long, with a non-slippery surface 20 cm above the base (trip plate). This rod is divided into four equal sections by five discs (25 cm in diameter), which enables four mice to walk on the rod at the same time. In the present study, the accelerating rotor mode was used (10-grade speeds from 2 to 20 r.p.m. for 5 min). The performance time was recorded while mice were running on the rod.
All data are presented as means ± standard error of the mean (SEM). The significance of differences was determined by one-way analysis of variance (ANOVA). Further statistical analysis for post hoc comparisons was performed using the Bonferroni/Dunn tests (StatView; Abacus Concepts, Berkeley, CA, USA). On the other hand, the significance of difference in rotation numbers/5 min and that of difference in areas of survival neurons in 6-OHDA-injected rats and MCAO-ischemic rats were determined by Student's t-test for single comparisons. Endurance performance (percentage of mice remaining on the rota-rod) was calculated by the Kaplan-Meier method. The statistical significance of differences was analyzed by the log-rank (Mantel-Cox) test.