Background
Parkinson's disease (PD) is a chronic neurodegenerative disease caused by dopaminergic cell death, and genetic and environmental factors are thought to affect the onset of PD. Cerebral infarction and stroke are acute neurodegenerative diseases caused by ischemic injury. Onsets of these diseases are thought be induced at least by oxidative stress, but the precise mechanisms are still not known. Although a precursor of dopamine, inhibitors of dopamine degradation and dopamine releasers have been used for PD therapy and an anti-oxidant have been used for cerebral infarction and stroke, cell death progresses during treatment. Identification of compounds or proteins that inhibit oxidative stress-induced neuronal cell death is necessary.
DJ-1 was first identified by our group as a novel oncogene product [
1] and later found to be a causative gene product of a familial form of PD, PARK7 [
2]. DJ-1 plays roles in transcriptional regulation [
3‐
9] and anti-oxidative stress reaction [
10‐
13], and loss of its function is thought to result in the onset of PD. DJ-1 has three cysteines at amino acid numbers 46, 53, and 106 (C46, C53, and C106, respectively). Although oxidation of C106 is necessary for DJ-1 to exert its activity [
12‐
15], further oxidation of C106 is thought to render DJ-1 inactive [
16,
17], and such oxidized DJ-1 has been observed in patients with the sporadic form of PD and Alzheimer disease [
18,
19].
We have shown that administration of DJ-1 protein dramatically reduced dopaminergic cell death and restored locomotion defect in PD model rats into which 6-hydroxydopamine (6-OHDA) had been injected [
20] and that intrastriatal injection of DJ-1 markedly reduced infarct size in cerebral ischemia in rats [
21], suggesting that DJ-1 is a pharmaceutical target for PD and cerebral ischemia. Another group also reported protective activity of DJ-1 against stroke [
22]. Furthermore, we identified compounds that bind to the C106 region of DJ-1, and these compounds including compounds A and B, like DJ-1 protein, prevented oxidative stress-induced dopaminergic cell death and restored locomotion defect in PD model rats and also reduced infarct size in cerebral ischemia in rats [
23‐
25]. These compounds were found by screening the University Compound library, which contains approximately 30,000 compounds.
In this study, we further screened DJ-1-binding compounds from the Zinc compound library that contains approximately 2,500,000 compounds. Of the compounds identified, compound-23 (comp-23) protected oxidative stress-induced cell death both in cultured cells and in PD and ischemia model rats and mice, and the protective activity of comp-23 seemed to be stronger than that of compound B.
Discussion
In this study, we identified a new DJ-1-binding compound, compound-23 (comp-23), from the Zinc compound library, and we found that comp-23 prevented oxidative stress-induced cell death both in cultured cells and in PD and ischemia model rats and mice. Comp-23 prevented cell death even at a high concentration of H2O2, a condition in which DJ-1-binding compound B did not show protective activity against cell death, suggesting that activity of comp-23 is stronger than that of compound B at least in cultured cells. Structures of comp-23 and comp-B appear similar at a glance but are clearly different, especially in the position of an amino group and benzene ring. Since the X-ray co-crystal structure of DJ-1 with compound B has not yet been elucidated, an exact binding structure of compound B within DJ-1 is not known at present. Determination of the structure-activity relationships between DJ-1 and DJ-1-binding compounds will be necessary to establish DJ-1-binding compounds that are more effective than compounds B and 23. The Zinc compound library used in this study is freely available. If other libraries are used for screening of DJ-1-binding compounds, novel compounds might be obtained.
Although comp-23 lacks direct scavenging activity against
.OH (Figure
5), comp-23 protected SH-SY5Y cells and primary rat neurons from oxidative stress-induced cell death (Figures
1,
2, and
3). Since comp-23 did not show a protective effect against oxidative stress-induced cell death in DJ-1-knockdown SH-SY5Y cells (Figure
4), comp-23 works in a DJ-1-dependent manner. Since a residual amount of DJ-1 was still expressed in DJ-1-knockdown SH-SY5Y cells, no protective activity of comp-23 in DJ-1-knockdown cells suggests that there is a threshold amount of DJ-1 for DJ-1-binding compounds to function in cells. Comp-23 prevented dopaminergic cell death both in the substantia nigra and striatum in 6-OHDA-administered PD model rats, resulting in suppression of locomotion defect of rats (Figures
7,
8,
9,
10,
11). Since a precursor of dopamine, inhibitors of dopamine degradation and dopamine releasers are used for PD therapy at present and since these drugs are used for symptomatic therapy, cell death progresses during treatment. In the present study, the intraperitoneal injection of comp-23 at before and after MCAO induced neuroprotection in a dose-dependent manner (Figure
11), and peripheral administration of comp-23 for 56 days prevented rotenone-induced Parkinsonian motor deficit (Figure
12). Based on these observations, we consider that comp-23 binds to endogenous DJ-1 protein after passing through the BBB and that this DJ-1-comp-23 complex shows the neuroprotective effect against ROS-mediated dopaminergic neurodegeneration. Thus, there is a possibility that chronic peripheral administration of comp-23 delays the progression of motor dysfunction in PD and/or brain stroke.
Comp-23 is not a simple anti-oxidant (Figure
5) and prevented excess oxidation of DJ-1 in cells that had been treated with various amounts of H
2O
2 (Figure
6A). Since excess oxidation of DJ-1 renders DJ-1 inactive, it is thought that comp-23 activates DJ-1 or maintains active forms of DJ-1, thereby affecting downstream targets of DJ-1. DJ-1, for instance, activates Nrf2, a master transcription factor of redox-related genes, by sequestering Keap1, a negative factor of Nrf2 [
32], and also activates the PI3 kinase/AKT pathway by inhibiting PTEN, a negative effecter of the PI3 kinase/AKT pathway, through direct binding with PTEN [
22,
33,
34]. Screening strategy is to identify compounds that bind to weakly oxidized DJ-1 with an SO
2H form of C106 using a model of such an oxidized DJ-1. Since reduced DJ-1 and oxidized DJ-1 are unable to be separately purified due to technical problem at present, we are not able to determine which form of DJ-1 is bound by comp-23. In vitro binding assays showed that comp-23 bound to recombinant DJ-1 that contains equal molar ratio of reduced and oxidized DJ-1 (Figure
1C), suggesting that comp-23 binds to both reduced DJ-1 and oxidized DJ-1. Furthermore, we examined dimer formation of DJ-1 in the presence and absent of comp-23. The results showed that comp-23 did not affect dimer formation of DJ-1 (Figure
6B). Since DJ-1 works as dimer, it is thought that dimer DJ-1 complexed with comp-23 shows protective activity against oxidative stress-induced neurodegeneration.
Reactive oxygen species are massively produced in the brain after cerebral ischemia and reperfusion. The antioxidant edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used as a brain protectant for stroke therapy and is effective within 24 hours after onset of stroke. It has been reported that DJ-1 immunoreactivity in human brain astrocytes is dependent on infarct presence and infarct age [
35], that DJ-1 is expressed in motor neurons after transient spinal cord ischemia in rabbits [
36] and that loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia [
27]. We and other group have reported that injection of DJ-1 or infection of DJ-1-containing virus reduced infarct size in cerebral ischemia in rats [
21,
22]. Furthermore, we have shown that administration of DJ-1-binding compound B also reduced infarct size of cerebral ischemia in rats [
24]. It is therefore thought that, like a PD model, comp-23 maintains activated forms of DJ-1 to activate Nrf2 and the AKT pathway, leading to reduction of ROS and to promotion of cell growth in ischemia model rats.
Methods
Materials
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).
Cell culture
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 [
17].
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 SO
2H form was obtained from a web site (
http://www.rcsb.org/pdb/). To obtain the structure of DJ-1 containing H
2O, 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 [
23].
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 [
37]. 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 × 10
6 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.
Isoelectric focusing
SH-SY5Y cells were incubated with 1 μM compound-23 or compound-B for 24 hours and then treated with various amounts of H
2O
2 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 [
10].
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.
ESR spectrometry
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.
Hemiparkinsonian rats
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 [
38]. 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 [
21]. 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.
Statistical evaluation
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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HA and SMMI-A conceptualized the study; YK, SW, MT, KT, TK, KT-N and HY carried out experiments; HM conducted the analyses and YK and HA wrote the manuscript. All authors read and approved the final manuscript.