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MG-132-induced neurotoxicity in rats
Brain Research Bulletin 88 (2012) 609–616
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Research report
DJ-1 protein protects dopaminergic neurons against 6-OHDA/MG-132-induced neurotoxicity in rats Shuang-Yong Sun a,b , Chun-Na An a,b , Xiao-Ping Pu a,b,∗ a b
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking Universit, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 15 April 2012 Received in revised form 23 May 2012 Accepted 24 May 2012 Available online 1 June 2012 Keywords: DJ-1 protein Parkinson’s disease 6-OHDA MG-132 SOD2 ␣-Synuclein
a b s t r a c t Parkinson disease (PD) is the second most common neurodegenerative disease, and it cannot be completely cured by current medications. In this study, DJ-1 protein was administrated into medial forebrain bundle of PD model rats those had been microinjected with 6-hydroxydopamine (6-OHDA) or MG-132. We found that DJ-1 protein could reduce apomorphine-induced rotations, inhibit reduction of dopamine contents and tyrosine hydroxylase levels in the striatum, and decrease dopaminergic neuron death in the substantia nigra. In 6-OHDA lesioned rats, uncoupling protein-4, uncoupling protein-5 and superoxide dismutase-2 (SOD2) mRNA and SOD2 protein were increased when DJ-1 protein was co-injected. Simultaneously, administration of DJ-1 protein reduced ␣-synuclein and hypoxia-inducible factor 1␣ mRNA and ␣-synuclein protein in MG-132 lesioned rats. Therefore, DJ-1 protein protected dopaminergic neurons in two PD model rats by increasing antioxidant capacity and inhibiting ␣-synuclein expression. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Parkinson’ disease (PD) is a slowly progressive neurodegenerative disorder characterized clinically by bradykinesia, rigidity, tremor, gait dysfunction, and postural instability (AcunaCastroviejo et al., 2011; Gandhi et al., 2009; Lev et al., 2009). It is caused by loss of dopaminergic neurons in the substantia nigra (SN), which results in decreased dopaminergic availability in the striatum (Guo et al., 2010). Current medications only provide symptomatic relief and fail to halt the death of dopaminergic neurons (Thomas and Beal, 2007; Yin et al., 2011). Therefore, new therapy needs to be developed to improve survival of dopaminergic neurons. Previous studies have shown that overexpression of wild-type (WT) DJ-1 could protect dopaminergic neurons from oxidative stress, reduce intracellular reactive oxygen species (ROS), and inhibit mutant human ␣-synuclein protein aggregation (Lev et al., 2009; Liu et al., 2008; Tang et al., 2006; Zhou and Freed, 2005). Up-regulating the DJ-1 protein by histone deacetylase inhibitors protected dopaminergic neurons against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity, reduced ␣-synuclein
aggregation in brain and prevented age-related deterioration in motor and cognitive function in a transgenic mouse model (Zhou et al., 2011). When DJ-1 protein was added to SH-SY5Y cells and mesencephalic neurons, production of ROS and cell death induced by 6-hydroxydopamine (6-OHDA) decreased obviously. In in vivo experiments, administered DJ-1 protein could be absorbed by dopaminergic neurons and remain stable for more than 24 h. DJ-1 protein could improve phenotypes of PD rats administrated with 6OHDA, while L166P DJ-1 (mutant form of DJ-1 found in PD patients) failed (Inden et al., 2006). Since DJ-1 protein could both antagonize oxidative stress and eliminate irregular protein aggregates, we hypothesized that administration of DJ-1 protein into the medial forebrain bundle (MFB) of PD model rats may provide protection from different toxins insults. Two toxins, 6-OHDA and MG-132, with different action pathways were used to prepare PD rat models. In this study, 6OHDA or MG-132 was microinjected into the MFB of rats in the presence of DJ-1 protein and results showed that DJ-1 protein dramatically improved movement dysfunction, recovered DA contents and tyrosine hydroxylase (TH) levels in the striatum and inhibited dopaminergic neuronal death in the SN. 2. Materials and methods
∗ Corresponding author at: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Science, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, PR China. Tel.: +86 10 8280 2431; fax: +86 10 8280 2431. E-mail address: [email protected] (X.-P. Pu). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.05.013
2.1. Animals and materials Sixty adult male Sprague-Dawley rats weighing 250–300 g were purchased from the Department of Laboratory Animal Science of Peking University Health Science Center and met the approval of the local animal committee, with the confirmation
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Table 1 Primer sequences used in real-time PCR. Primer
GenBank accession number
Primer sequence
Amplicon (bp)
UCP4
NM053500
F: 5 -ATTATCCCCTCTGGAAGTCCG-3 R: 5 -ACTCCACGGAATCTCAAGGGT-3
148
UCP5
NM001166450
F: 5 -CGAGGAACTGGCAAGAT-3 R: 5 -GCAACAATAGAGGCAAGG-3
196
SOD2
NM017051
F: 5 -GGCTAAGGATGGATGGAGTGG-3 R: 5 -GCACAATGTCACTCCTCTCCG-3
140
␣-Synuclein
NM009221
F: 5 -ATCCTGGCAGTGAGGCTTATG-3 R: 5 -TGACTGGGCACATTGGAACTG-3
151
HIF-1␣
NM024359
F: 5 -TCATATCACTGGACTTCGGCAG-3 R: 5 -GAAGTGGCTTTGGAGTTTCAGAG-3
162
-Actin
NM031144
F: 5 -GTGACGTTGACATCCGTAAAGAC-3 R: 5 -GCCAGGATAGAGCCACCAAT-3
187
number, SCXK (Jing) 2006–0008. Rats were maintained under standard room temperature (22 ± 2 ◦ C) and relative humidity (60% ± 10%) with a 12 h light/dark cycle. Rats were allowed free access to food and water ad libitum throughout the acclimatization and experimental period. All experiments were performed under the guidelines of the Experimental Laboratory Animal Committee of Peking University Health Science Center and were in strict accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 6-OHDA and MG-132 were purchased from Sigma company (Sigma–Aldrich, St. Louis, USA). Apomorphine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other reagents or drugs were of analytical grade. 2.2. DJ-1 protein expression and purification DJ-1 protein was expressed in and purified from Escherichia coli as described previously (Nagakubo et al., 1997; Taira et al., 2001). It was stored at −80 ◦ C until used. 2.3. Creation of PD rat model with 6-OHDA or MG-132 lesioning Rats were randomly divided into the following five groups: control group, 6OHDA group, 6-OHDA + DJ-1 group, MG-132 group and MG-132 + DJ-1 group. For stereotaxic microinjection, rats were anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) and immobilized in a stereotaxic frame. Rats of 6-OHDA group and MG-132 group were injected with 6-OHDA (6.0 g, final concentration 6 mM) or MG-132 (1.9 g, final concentration 1 mM) in a final volume of 4 l of phosphatebuffered saline (PBS), while rats of control group were injected with same volume of PBS. Rats of 6-OHDA + DJ-1 group and MG-132 + DJ-1 group were injected with the same dose of 6-OHDA or MG-132 in the presence of DJ-1 protein (7.7 g, final concentration 80 M). Stereotaxic coordinates, relative to bregma, were: AP = −4.8 mm, ML = −1.8 mm, DV = −7.8 mm, and the toothbar was set at −2.4 mm. Injection speed was 1.0 l/min and the syringe was kept in place for an additional 3 min before it was slowly retracted.
for 2 days. A series of 20 m thick coronal sections were cut through the ventral mesencephalon using a cryostat (Leica, Nussloch, Germany). TH-immunohistochemistry on SN sections was performed using previously published 3,3 -diaminobenzidine (DAB) protocols (Chen et al., 2011; Geng et al., 2004; Zhang et al., 2010). The microphotographs were then captured with a BX-50 microscope (Olympus, Tokyo Japan) and the immunohistochemistry results were analyzed using Image-Pro Plus 6.0 Software.
2.7. Real-time PCR Total RNA in the right SN was extracted using the TRIZOL® Reagent (invitrogen, MD, USA) and it was reverse-transcribed using the RevertAidTM First Strand cDNA Synthesis kit (Fermentas China, Beijing, China) according to manufacturer’s procedures. The reactions were incubated for 5 min at 25 ◦ C, 60 min at 42 ◦ C, heated for 5 min at 70 ◦ C and placed at 4 ◦ C. The cycling profile involved PCR activation step at 50 ◦ C for 2 min and at 95 ◦ C for 10 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, primer annealing at 60 ◦ C for 20 s, and extension at 72 ◦ C for 30 s. The fluorescence was measured at the end of each cycle. Primer sequences used saw Table 1. An analysis of relative gene expression data was performed, using the 2−CT method on an ABI PRISM® 7500 Sequence Detection System Software (Applied Biosystems, CA, USA). The fold change in studied gene expression, normalized to control, was calculated using: relative fold increase = 2−CT .
2.4. Apomorphine-induced rotational behavior Four weeks postlesion, rats of each group received a single intraperitoneal injection of apomorphine (1 mg/kg in normal saline). Rotational asymmetry was scored for 30 min continuously and complete contralateral rotations were scored. One day after behavioral tests, rats were sacrificed and required tissues were obtained for subsequent experiments. 2.5. DA and its metabolites measurements by HPLC Six rats from each group were sacrificed and their brains were quickly removed and placed on ice. The right striatums were dissected out and weighted. The contents of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). HPLC analysis was performed based on methods described previously (Geng et al., 2004). 2.6. Immunohistochemistry Three rats from each group were perfused through the aorta with PBS followed by cold 4% paraformaldehyde, under deep anesthesia with pentobarbital (100 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with 4% paraformaldehyde and then transferred to 30% sucrose solution at 4 ◦ C
Fig. 1. Cell lysates containing GST-DJ-1 protein and DJ-1 protein without GST label electrophoresised by SDS-PAGE. (A) SDS-PAGE of cell lysates containing GST-DJ-1 protein (M: Protein Marker; C: cell lysates of E. coli BL21 containing GST-DJ-1 protein before induction by IPTG; 1–5: cell lysates of E. coli BL21 containing GST-DJ-1 protein after induction by IPTG). (B) SDS-PAGE of DJ-1 protein without GST label (M: Protein Marker; 1–7: DJ-1 protein released from glutathione-agarose beads after cutting by Prescission Protease).
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Fig. 2. Effects of DJ-1 protein on apomorphine-induced rotations and DA and its metabolites reduction in the striatum. (A) Apomorphine-induced rotations (n = 12). (B) Chromatograms of each group. (C) DA, DOPAC and HVA contents in the right striatum of each group (n = 6). Data are expressed as means ± SD. **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. 6-OHDA group or MG-132 group. 2.8. Western blot Protein of the right striatum was obtained and assayed based on methods described previously (Li et al., 2009; Yang and Pu, 2009). Equal amount of proteins for each group were separated in a 12.5% SDS-PAGE gel and electrophoretically transferred to PVDF (Millipore, MA, USA), blocked, and probed overnight at 4 ◦ C with primary antibody. The following antibodies were used: rabbit polyclonal anti-TH (1:1000), mouse monoclonal anti-superoxide dismutase-2 (SOD2, 1:500), mouse monoclonal anti-␣-synuclein (1:500), rabbit polyclonal anti--actin (1:500). The membranes were incubated with the corresponding secondary antibody labeled with horseradish peroxidase (1:2000) for 2 h at 37 ◦ C. The protein bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and analyzed by Image-Pro Plus 6.0 Software. 2.9. Statistics Data were collected from at least three independent experiments. Statistics were performed with original data via paired t-test or with the data normalized by control via a one-way ANOVA. A P-value less than 0.05 was considered as significant difference.
3. Results 3.1. Expression and purification of DJ-1 protein The cell lysates and purified DJ-1 protein were separated by 12.5% SDS-PAGE and stained with Coomassie brilliant blue. On the SDS-PAGE of cell lysates, clear bands were detected at about 50 kDa, the predicted size of the GST-DJ-1 protein (Fig. 1A). After GST tag
was removed with Prescission Protease, DJ-1 protein was visualized at about 24 kDa, which showed that DJ-1 protein was successfully released from the GST-DJ-1 fusion protein (Fig. 1B). 3.2. DJ-1 protein reduced apomorphine-induced rotation At four weeks postlesion, rats of each group were assessed for rotational behavior (Fig. 2A). Apomorphine injection caused marked turning in 6-OHDA group and MG-132 group rats (both P < 0.01 vs. control group), which was similar to previous reports (Ahmad et al., 2005; Baluchnejadmojarad et al., 2009; Chen et al., 2003). Rotations of 6-OHDA + DJ-1 group and MG-132 + DJ-1 group were seen to decrease significantly (P < 0.01 and P < 0.05 vs. 6-OHDA group or MG-132 group, respectively). 3.3. DJ-1 protein restrained DA contents reduction DA and its metabolites contents in the right striatum were determined by HPLC-ECD at four weeks postlesion (Fig. 2B and C). DA, DOPAC and HVA contents in the right striatum of 6-OHDA-lesioned rats were 78.0%, 48.2% and 40.9% lower than those in control group rats (all P < 0.01). After administration of DJ-1 protein, the striatal DA content of 6-OHDA + DJ-1 group was higher than that in 6-OHDA group rats (P < 0.01), and the striatal DOPAC and HVA contents had no statistically significant increase. Simultaneously,
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Fig. 3. Effects of DJ-1 protein on dopaminergic neurons death in the SN and TH levels reduction in the striatum. (A) TH immunohistochemistry of the right SN. (B) TH-positive neurons count. (C–E) TH contents in the right striatum detected by Western blot. Data are expressed as means (% of control) ± SD (n = 3). **P < 0.01 vs. control group; ## P < 0.01 vs. 6-OHDA group or MG-132 group. Solid line scale bar = 200 m; dotted line scale bar = 100 m.
DA, DOPAC and HVA contents in the right striatum of MG-132lesioned rats were 62.6%, 65.5% and 56.6% lower than those in control group rats (all P < 0.01). After administration of DJ-1 protein, the right striatal DA and DOPAC contents of MG-132 + DJ-1 group were higher than those of the MG-132 group (P < 0.01 or P < 0.05), and the right striatal HVA content had no statistically significant increase. We concluded that DJ-1 protein inhibited the reduction of DA contents in the right striatum induced by 6-OHDA or MG-132 apparently. 3.4. DJ-1 protein inhibited dopaminergic neuronal death in the SN and TH levels reduction in the striatum The expression of TH in the SN was measured by immunohistochemistry (Fig. 3 A, B). Through analysis of TH-positive neurons in
the right SN, the 6-OHDA group and MG-132 group showed significant losses of dopaminergic neurons (P < 0.01 vs. control group). Loss of dopaminergic neurons was significantly inhibited by simultaneous administration of DJ-1 protein (both P < 0.01 vs. 6-OHDA group or MG-132 group, respectively). Further, TH protein levels in the right striatal tissues were measured by Western blot (Fig. 3C–E). In 6-OHDA group and MG-132 group, TH protein levels reduced significantly (both P < 0.01 vs. control group). DJ-1 protein treatment increased TH protein levels in the right striatum of 6-OHDA + DJ-1 group and MG-132 + DJ1 group obviously (both P < 0.01 vs. 6-OHDA group or MG-132 group, respectively). These data indicated that DJ-1 protein could protect dopaminergic neurons in the SN and decreased reduction of TH expression in the striatum from 6-OHDA or MG-132 toxicity.
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Fig. 4. DJ-1 protein improved antioxidant capacity of dopaminergic neurons in the SN and striatum. (A–C) Levels of UCP4, UCP5 and SOD2 mRNA in the right SN of 6-OHDA lesioned rats detected by real-time PCR. (D and E) SOD2 protein expression in the right striatum of 6-OHDA lesioned rats detected by Western blot. Data are expressed as means ± SD or means (% of control) ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. 6-OHDA group.
3.5. DJ-1 protein increased antioxidant capacity in dopaminergic neurons
3.6. DJ-1 protein reduced ˛-synuclein expression and HIF-1˛ mRNA expression in dopaminergic neurons
In order to explore the mechanisms by which DJ-1 protein protected the dopaminergic neurons, real-time PCR was used to measure the mRNA levels of uncoupling protein-4 (UCP4), uncoupling protein-5 (UCP5) and SOD2 in the right SN of the 6-OHDA lesioned rats (Fig. 4A–C). Levels of UCP4 and UCP5 mRNA in the right SN of 6-OHDA group decreased (both P < 0.05 vs. control group) while that of SOD2 manifested significant elevated (P < 0.05 vs. control group). Data showed that simultaneous microinjection of DJ-1 protein increased UCP4, UCP5 and SOD2 mRNA expression in the SN compared with microinjection of 6-OHDA alone (P < 0.05 or P < 0.01). Subsequently, the level of SOD2 protein in the right striatum of 6-OHDA lesioned rats was detected by Western blot (Fig. 4D and E). After 6-OHDA lesion, SOD2 protein manifested obvious increase compared with control group (P < 0.01). Microinjection of DJ-1 protein was found to increase SOD2 protein expression compared with microinjection of 6-OHDA alone (P < 0.05).
The levels of ␣-synuclein and hypoxia-inducible factor 1␣ (HIF1␣) mRNA in the right SN of the MG-132 lesioned rats were detected using real-time PCR (Fig. 5A and B). Microinjection of MG-132 significantly increased ␣-synuclein and HIF-1␣ mRNA expression compared with control group (both P < 0.01). Administration of DJ1 protein was found to depress ␣-synuclein and HIF-1␣ mRNA expression apparently (both P < 0.01 vs. MG-132 group). Similar to mRNA results, ␣-synuclein protein expression in the right striatum of MG-132 group increased significantly compared with control group (P < 0.01), and DJ-1 protein obviously reduced ␣-synuclein protein expression in MG-132 + DJ-1 group (P < 0.01 vs. MG-132 group) (Fig. 5C and D). 4. Discussion In the present study, DJ-1 protein was microinjected into the MFB of PD rats those had been injected to 6-OHDA or MG-132.
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Fig. 5. DJ-1 protein inhibited ␣-synuclein expression and HIF-1␣ mRNA expression in the SN and striatum of dopaminergic neurons. (A and B) Levels of HIF-1␣ and ␣synuclein mRNA in the right SN of MG-132 lesioned rats detected by real-time PCR. (C and D) ␣-Synuclein protein expression in the right striatum of MG-132 lesioned rats detected by Western blot. Data are expressed as means ± SD or means (% of control) ± SD (n = 3). **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. MG-132 group.
Administration of DJ-1 protein apparently improved PD phenotypes of rats, including improving motor abnormality, inhibiting reduction of DA contents and TH levels in the striatum, and decreasing dopaminergic neuron death in the SN. Furthermore, by measuring a series of mRNA and protein in the SN and striatum, DJ-1 protein was found to protect dopaminergic neurons through diverse pathways. Traditional animal models of PD are based on the use of toxins, which selectively accumulate in the SN dopaminergic neurons, causing cellular dys-function and death, and 6-OHDA is the most frequently used one (Beal, 2010). In this experiment, results of 6OHDA lesiond rat model were consistent with previous reports (Baluchnejadmojarad et al., 2009; Inden et al., 2006). On the other hand, impairment of the ubiquitin–proteasome system (UPS) has been reported to play an important role in the pathogenesis of PD (Bedford et al., 2008). Microinjection with proteasome inhibitor MG-132 into the MFB or SN of C57BL/6 mice induced a sustained dopaminergic neurons degeneration, which recapitulated many neuropathological and behavioral features of PD (Sun et al., 2006; Xie et al., 2010). In our study, MG-132 was microinjected into the right MFB of rats, which induced movement disorders, DA and its metabolites contents and TH levels reduction in the striatum, and dopaminergic neuron degeneration in the SN. Microinjection of 6-OHDA into the MFB of rats leads to oxidative stimuli and generates ROS, which causes damage to mitochondria in dopaminergic neurons. UCPs are a class of mitochondrial ion channel, whose open probability is increased by superoxide (Krauss et al., 2005). Blocking UCPs may cause superoxide concentrations and oxidation of mitochondrial proteins to increase. Guzman et al. confirmed that knocking out DJ-1 downregulated the expression of UCPs (UCP4 and UCP5) and increased oxidation of matrix
proteins specifically in substantia nigra compacta (SNc) of dopaminergic neurons (Guzman et al., 2010). In our study, administration of DJ-1 protein increased the abundance of UCP4 and UCP5 mRNAs in the SN and thus might enhance mitochondrial uncoupling in response to oxidant stress, which made a significant contribution to maintain the stability of mitochondria and preserve dopaminergic neuron survival. In addition to UCPs, SOD2 also plays an important role when mitochondria suffers damage from oxidative stress (Guzman et al., 2010). SODs are a ubiquitous family of enzymes those function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian SODs have been biochemically and molecularly characterized. SOD2 is one of the three SODs located in the mitochondria and serves as the primary defense against mitochondrial superoxide (Lebovitz et al., 1996; Zelko et al., 2002). SOD2 mutation may increase PD risk especially for people who are frequently exposured to pesticides (Fong et al., 2007). The expression of human SOD2 could be induced by DJ-1 through increasing the activity of peroxisome proliferatoractivated receptor-␥ co-activator 1␣ (PGC-1␣) (Zhong and Xu, 2008). In our study, SOD2 mRNA and protein in the SN and striatum of PD model rats increased obviously after administration of DJ-1 protein into the MFB. When oxidative stress signals overwhelmed the ROS defense machinery after 6-OHDA administration, DJ-1 protein led to increased synergistic activation of the SOD2, thus slowing down the oxidative damage. mRNA and protein of SOD2 in rats of 6-OHDA group were also found to increase, which was consistent with previous reports (Carange et al., 2011; LahaieCollins et al., 2008). The reason for this phenomenon might be that SOD2 was increased by 6-OHDA induced superoxide ions under a response-to-stress mechanism. Therefore, we concluded that
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Hiroyoshi Ariga of Hokkaido University for providing the pGEX-6p1-GST DJ-1 plasmid.
References
Fig. 6. Proposed mechanisms by which DJ-1 protein protects against 6-OHDA and MG-132 toxicity.
DJ-1 protein increased antioxidant capacity to protect dopaminergic neurons from 6-OHDA damage. MG-132 is different with 6-OHDA in action mechanism, and it mainly results in irregular protein aggregation in the brain. Xie et al. certificated that microinjection with MG-132 into the MFB of mice led to sustained dopaminergic neuron degeneration with ␣-synuclein aggregation (Xie et al., 2010). ␣-Synuclein resided predominantly in the ‘presynaptic’ or output terminals of neurons, and stuck-together ␣-synuclein proteins constituted the tiny fibrils making up Lewy bodies, which were thought to be neuronal losses and pathological signs of PD (Schnabel, 2010). In our study, ␣-synulcein mRNA and protein were found to increase in MG-132 lesioned rats. Microinjection of DJ-1 protein obviously decreased ␣-synulcein at both protein and mRNA levels. Therefore, DJ-1 protein might protect dopaminergic neurons in the SN and striatum through reducing ␣-synulcein protein expression. Furthermore, we also found that HIF-1␣ mRNA in the dopaminergic neurons increased after administration of MG-132. HIF-1␣ is the only transcription factor associated with down-regulation of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrphic factor (GDNF), a neuroprotective molecule for dopaminergic neurons (Foti et al., 2010). HIF-1␣ overexpression may cause RET down-regulation and lead to loss of GDNF protection in dopaminergic neurons. Administration of DJ1 protein decreased HIF-1␣ mRNA expression and thus might repress down-regulation of RET, in which case ligand of RET, GDNF might promote survival of dopaminergic neurons in PD. Further study was needed to investigate molecular mechanism underlying this pathway. Therefore, we concluded that DJ-1 protein inhibited ␣-synulcein protein expression to protect dopaminergic neurons from MG-132 damage (Fig. 6). In summary, two toxins with different action mechanisms were used to modeling PD in rats. Simultaneous administration of DJ-1 protein into the MFB of rats dramatically improved PD phenotypes in two models. Besides, we also found that DJ-1 protein protected dopaminergic neurons through different pathways, including increasing antioxidant capacity and inhibiting ␣-synuclein expression. However, detailed molecular mechanisms were needed to be investigated in further study. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by the Doctor Fund of the Ministry of Education in China (20090001110069). We thank Professor
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Research report
DJ-1 protein protects dopaminergic neurons against 6-OHDA/MG-132-induced neurotoxicity in rats Shuang-Yong Sun a,b , Chun-Na An a,b , Xiao-Ping Pu a,b,∗ a b
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking Universit, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 15 April 2012 Received in revised form 23 May 2012 Accepted 24 May 2012 Available online 1 June 2012 Keywords: DJ-1 protein Parkinson’s disease 6-OHDA MG-132 SOD2 ␣-Synuclein
a b s t r a c t Parkinson disease (PD) is the second most common neurodegenerative disease, and it cannot be completely cured by current medications. In this study, DJ-1 protein was administrated into medial forebrain bundle of PD model rats those had been microinjected with 6-hydroxydopamine (6-OHDA) or MG-132. We found that DJ-1 protein could reduce apomorphine-induced rotations, inhibit reduction of dopamine contents and tyrosine hydroxylase levels in the striatum, and decrease dopaminergic neuron death in the substantia nigra. In 6-OHDA lesioned rats, uncoupling protein-4, uncoupling protein-5 and superoxide dismutase-2 (SOD2) mRNA and SOD2 protein were increased when DJ-1 protein was co-injected. Simultaneously, administration of DJ-1 protein reduced ␣-synuclein and hypoxia-inducible factor 1␣ mRNA and ␣-synuclein protein in MG-132 lesioned rats. Therefore, DJ-1 protein protected dopaminergic neurons in two PD model rats by increasing antioxidant capacity and inhibiting ␣-synuclein expression. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Parkinson’ disease (PD) is a slowly progressive neurodegenerative disorder characterized clinically by bradykinesia, rigidity, tremor, gait dysfunction, and postural instability (AcunaCastroviejo et al., 2011; Gandhi et al., 2009; Lev et al., 2009). It is caused by loss of dopaminergic neurons in the substantia nigra (SN), which results in decreased dopaminergic availability in the striatum (Guo et al., 2010). Current medications only provide symptomatic relief and fail to halt the death of dopaminergic neurons (Thomas and Beal, 2007; Yin et al., 2011). Therefore, new therapy needs to be developed to improve survival of dopaminergic neurons. Previous studies have shown that overexpression of wild-type (WT) DJ-1 could protect dopaminergic neurons from oxidative stress, reduce intracellular reactive oxygen species (ROS), and inhibit mutant human ␣-synuclein protein aggregation (Lev et al., 2009; Liu et al., 2008; Tang et al., 2006; Zhou and Freed, 2005). Up-regulating the DJ-1 protein by histone deacetylase inhibitors protected dopaminergic neurons against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity, reduced ␣-synuclein
aggregation in brain and prevented age-related deterioration in motor and cognitive function in a transgenic mouse model (Zhou et al., 2011). When DJ-1 protein was added to SH-SY5Y cells and mesencephalic neurons, production of ROS and cell death induced by 6-hydroxydopamine (6-OHDA) decreased obviously. In in vivo experiments, administered DJ-1 protein could be absorbed by dopaminergic neurons and remain stable for more than 24 h. DJ-1 protein could improve phenotypes of PD rats administrated with 6OHDA, while L166P DJ-1 (mutant form of DJ-1 found in PD patients) failed (Inden et al., 2006). Since DJ-1 protein could both antagonize oxidative stress and eliminate irregular protein aggregates, we hypothesized that administration of DJ-1 protein into the medial forebrain bundle (MFB) of PD model rats may provide protection from different toxins insults. Two toxins, 6-OHDA and MG-132, with different action pathways were used to prepare PD rat models. In this study, 6OHDA or MG-132 was microinjected into the MFB of rats in the presence of DJ-1 protein and results showed that DJ-1 protein dramatically improved movement dysfunction, recovered DA contents and tyrosine hydroxylase (TH) levels in the striatum and inhibited dopaminergic neuronal death in the SN. 2. Materials and methods
∗ Corresponding author at: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Science, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, PR China. Tel.: +86 10 8280 2431; fax: +86 10 8280 2431. E-mail address: [email protected] (X.-P. Pu). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.05.013
2.1. Animals and materials Sixty adult male Sprague-Dawley rats weighing 250–300 g were purchased from the Department of Laboratory Animal Science of Peking University Health Science Center and met the approval of the local animal committee, with the confirmation
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Table 1 Primer sequences used in real-time PCR. Primer
GenBank accession number
Primer sequence
Amplicon (bp)
UCP4
NM053500
F: 5 -ATTATCCCCTCTGGAAGTCCG-3 R: 5 -ACTCCACGGAATCTCAAGGGT-3
148
UCP5
NM001166450
F: 5 -CGAGGAACTGGCAAGAT-3 R: 5 -GCAACAATAGAGGCAAGG-3
196
SOD2
NM017051
F: 5 -GGCTAAGGATGGATGGAGTGG-3 R: 5 -GCACAATGTCACTCCTCTCCG-3
140
␣-Synuclein
NM009221
F: 5 -ATCCTGGCAGTGAGGCTTATG-3 R: 5 -TGACTGGGCACATTGGAACTG-3
151
HIF-1␣
NM024359
F: 5 -TCATATCACTGGACTTCGGCAG-3 R: 5 -GAAGTGGCTTTGGAGTTTCAGAG-3
162
-Actin
NM031144
F: 5 -GTGACGTTGACATCCGTAAAGAC-3 R: 5 -GCCAGGATAGAGCCACCAAT-3
187
number, SCXK (Jing) 2006–0008. Rats were maintained under standard room temperature (22 ± 2 ◦ C) and relative humidity (60% ± 10%) with a 12 h light/dark cycle. Rats were allowed free access to food and water ad libitum throughout the acclimatization and experimental period. All experiments were performed under the guidelines of the Experimental Laboratory Animal Committee of Peking University Health Science Center and were in strict accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 6-OHDA and MG-132 were purchased from Sigma company (Sigma–Aldrich, St. Louis, USA). Apomorphine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other reagents or drugs were of analytical grade. 2.2. DJ-1 protein expression and purification DJ-1 protein was expressed in and purified from Escherichia coli as described previously (Nagakubo et al., 1997; Taira et al., 2001). It was stored at −80 ◦ C until used. 2.3. Creation of PD rat model with 6-OHDA or MG-132 lesioning Rats were randomly divided into the following five groups: control group, 6OHDA group, 6-OHDA + DJ-1 group, MG-132 group and MG-132 + DJ-1 group. For stereotaxic microinjection, rats were anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) and immobilized in a stereotaxic frame. Rats of 6-OHDA group and MG-132 group were injected with 6-OHDA (6.0 g, final concentration 6 mM) or MG-132 (1.9 g, final concentration 1 mM) in a final volume of 4 l of phosphatebuffered saline (PBS), while rats of control group were injected with same volume of PBS. Rats of 6-OHDA + DJ-1 group and MG-132 + DJ-1 group were injected with the same dose of 6-OHDA or MG-132 in the presence of DJ-1 protein (7.7 g, final concentration 80 M). Stereotaxic coordinates, relative to bregma, were: AP = −4.8 mm, ML = −1.8 mm, DV = −7.8 mm, and the toothbar was set at −2.4 mm. Injection speed was 1.0 l/min and the syringe was kept in place for an additional 3 min before it was slowly retracted.
for 2 days. A series of 20 m thick coronal sections were cut through the ventral mesencephalon using a cryostat (Leica, Nussloch, Germany). TH-immunohistochemistry on SN sections was performed using previously published 3,3 -diaminobenzidine (DAB) protocols (Chen et al., 2011; Geng et al., 2004; Zhang et al., 2010). The microphotographs were then captured with a BX-50 microscope (Olympus, Tokyo Japan) and the immunohistochemistry results were analyzed using Image-Pro Plus 6.0 Software.
2.7. Real-time PCR Total RNA in the right SN was extracted using the TRIZOL® Reagent (invitrogen, MD, USA) and it was reverse-transcribed using the RevertAidTM First Strand cDNA Synthesis kit (Fermentas China, Beijing, China) according to manufacturer’s procedures. The reactions were incubated for 5 min at 25 ◦ C, 60 min at 42 ◦ C, heated for 5 min at 70 ◦ C and placed at 4 ◦ C. The cycling profile involved PCR activation step at 50 ◦ C for 2 min and at 95 ◦ C for 10 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, primer annealing at 60 ◦ C for 20 s, and extension at 72 ◦ C for 30 s. The fluorescence was measured at the end of each cycle. Primer sequences used saw Table 1. An analysis of relative gene expression data was performed, using the 2−CT method on an ABI PRISM® 7500 Sequence Detection System Software (Applied Biosystems, CA, USA). The fold change in studied gene expression, normalized to control, was calculated using: relative fold increase = 2−CT .
2.4. Apomorphine-induced rotational behavior Four weeks postlesion, rats of each group received a single intraperitoneal injection of apomorphine (1 mg/kg in normal saline). Rotational asymmetry was scored for 30 min continuously and complete contralateral rotations were scored. One day after behavioral tests, rats were sacrificed and required tissues were obtained for subsequent experiments. 2.5. DA and its metabolites measurements by HPLC Six rats from each group were sacrificed and their brains were quickly removed and placed on ice. The right striatums were dissected out and weighted. The contents of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). HPLC analysis was performed based on methods described previously (Geng et al., 2004). 2.6. Immunohistochemistry Three rats from each group were perfused through the aorta with PBS followed by cold 4% paraformaldehyde, under deep anesthesia with pentobarbital (100 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with 4% paraformaldehyde and then transferred to 30% sucrose solution at 4 ◦ C
Fig. 1. Cell lysates containing GST-DJ-1 protein and DJ-1 protein without GST label electrophoresised by SDS-PAGE. (A) SDS-PAGE of cell lysates containing GST-DJ-1 protein (M: Protein Marker; C: cell lysates of E. coli BL21 containing GST-DJ-1 protein before induction by IPTG; 1–5: cell lysates of E. coli BL21 containing GST-DJ-1 protein after induction by IPTG). (B) SDS-PAGE of DJ-1 protein without GST label (M: Protein Marker; 1–7: DJ-1 protein released from glutathione-agarose beads after cutting by Prescission Protease).
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Fig. 2. Effects of DJ-1 protein on apomorphine-induced rotations and DA and its metabolites reduction in the striatum. (A) Apomorphine-induced rotations (n = 12). (B) Chromatograms of each group. (C) DA, DOPAC and HVA contents in the right striatum of each group (n = 6). Data are expressed as means ± SD. **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. 6-OHDA group or MG-132 group. 2.8. Western blot Protein of the right striatum was obtained and assayed based on methods described previously (Li et al., 2009; Yang and Pu, 2009). Equal amount of proteins for each group were separated in a 12.5% SDS-PAGE gel and electrophoretically transferred to PVDF (Millipore, MA, USA), blocked, and probed overnight at 4 ◦ C with primary antibody. The following antibodies were used: rabbit polyclonal anti-TH (1:1000), mouse monoclonal anti-superoxide dismutase-2 (SOD2, 1:500), mouse monoclonal anti-␣-synuclein (1:500), rabbit polyclonal anti--actin (1:500). The membranes were incubated with the corresponding secondary antibody labeled with horseradish peroxidase (1:2000) for 2 h at 37 ◦ C. The protein bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and analyzed by Image-Pro Plus 6.0 Software. 2.9. Statistics Data were collected from at least three independent experiments. Statistics were performed with original data via paired t-test or with the data normalized by control via a one-way ANOVA. A P-value less than 0.05 was considered as significant difference.
3. Results 3.1. Expression and purification of DJ-1 protein The cell lysates and purified DJ-1 protein were separated by 12.5% SDS-PAGE and stained with Coomassie brilliant blue. On the SDS-PAGE of cell lysates, clear bands were detected at about 50 kDa, the predicted size of the GST-DJ-1 protein (Fig. 1A). After GST tag
was removed with Prescission Protease, DJ-1 protein was visualized at about 24 kDa, which showed that DJ-1 protein was successfully released from the GST-DJ-1 fusion protein (Fig. 1B). 3.2. DJ-1 protein reduced apomorphine-induced rotation At four weeks postlesion, rats of each group were assessed for rotational behavior (Fig. 2A). Apomorphine injection caused marked turning in 6-OHDA group and MG-132 group rats (both P < 0.01 vs. control group), which was similar to previous reports (Ahmad et al., 2005; Baluchnejadmojarad et al., 2009; Chen et al., 2003). Rotations of 6-OHDA + DJ-1 group and MG-132 + DJ-1 group were seen to decrease significantly (P < 0.01 and P < 0.05 vs. 6-OHDA group or MG-132 group, respectively). 3.3. DJ-1 protein restrained DA contents reduction DA and its metabolites contents in the right striatum were determined by HPLC-ECD at four weeks postlesion (Fig. 2B and C). DA, DOPAC and HVA contents in the right striatum of 6-OHDA-lesioned rats were 78.0%, 48.2% and 40.9% lower than those in control group rats (all P < 0.01). After administration of DJ-1 protein, the striatal DA content of 6-OHDA + DJ-1 group was higher than that in 6-OHDA group rats (P < 0.01), and the striatal DOPAC and HVA contents had no statistically significant increase. Simultaneously,
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Fig. 3. Effects of DJ-1 protein on dopaminergic neurons death in the SN and TH levels reduction in the striatum. (A) TH immunohistochemistry of the right SN. (B) TH-positive neurons count. (C–E) TH contents in the right striatum detected by Western blot. Data are expressed as means (% of control) ± SD (n = 3). **P < 0.01 vs. control group; ## P < 0.01 vs. 6-OHDA group or MG-132 group. Solid line scale bar = 200 m; dotted line scale bar = 100 m.
DA, DOPAC and HVA contents in the right striatum of MG-132lesioned rats were 62.6%, 65.5% and 56.6% lower than those in control group rats (all P < 0.01). After administration of DJ-1 protein, the right striatal DA and DOPAC contents of MG-132 + DJ-1 group were higher than those of the MG-132 group (P < 0.01 or P < 0.05), and the right striatal HVA content had no statistically significant increase. We concluded that DJ-1 protein inhibited the reduction of DA contents in the right striatum induced by 6-OHDA or MG-132 apparently. 3.4. DJ-1 protein inhibited dopaminergic neuronal death in the SN and TH levels reduction in the striatum The expression of TH in the SN was measured by immunohistochemistry (Fig. 3 A, B). Through analysis of TH-positive neurons in
the right SN, the 6-OHDA group and MG-132 group showed significant losses of dopaminergic neurons (P < 0.01 vs. control group). Loss of dopaminergic neurons was significantly inhibited by simultaneous administration of DJ-1 protein (both P < 0.01 vs. 6-OHDA group or MG-132 group, respectively). Further, TH protein levels in the right striatal tissues were measured by Western blot (Fig. 3C–E). In 6-OHDA group and MG-132 group, TH protein levels reduced significantly (both P < 0.01 vs. control group). DJ-1 protein treatment increased TH protein levels in the right striatum of 6-OHDA + DJ-1 group and MG-132 + DJ1 group obviously (both P < 0.01 vs. 6-OHDA group or MG-132 group, respectively). These data indicated that DJ-1 protein could protect dopaminergic neurons in the SN and decreased reduction of TH expression in the striatum from 6-OHDA or MG-132 toxicity.
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Fig. 4. DJ-1 protein improved antioxidant capacity of dopaminergic neurons in the SN and striatum. (A–C) Levels of UCP4, UCP5 and SOD2 mRNA in the right SN of 6-OHDA lesioned rats detected by real-time PCR. (D and E) SOD2 protein expression in the right striatum of 6-OHDA lesioned rats detected by Western blot. Data are expressed as means ± SD or means (% of control) ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. 6-OHDA group.
3.5. DJ-1 protein increased antioxidant capacity in dopaminergic neurons
3.6. DJ-1 protein reduced ˛-synuclein expression and HIF-1˛ mRNA expression in dopaminergic neurons
In order to explore the mechanisms by which DJ-1 protein protected the dopaminergic neurons, real-time PCR was used to measure the mRNA levels of uncoupling protein-4 (UCP4), uncoupling protein-5 (UCP5) and SOD2 in the right SN of the 6-OHDA lesioned rats (Fig. 4A–C). Levels of UCP4 and UCP5 mRNA in the right SN of 6-OHDA group decreased (both P < 0.05 vs. control group) while that of SOD2 manifested significant elevated (P < 0.05 vs. control group). Data showed that simultaneous microinjection of DJ-1 protein increased UCP4, UCP5 and SOD2 mRNA expression in the SN compared with microinjection of 6-OHDA alone (P < 0.05 or P < 0.01). Subsequently, the level of SOD2 protein in the right striatum of 6-OHDA lesioned rats was detected by Western blot (Fig. 4D and E). After 6-OHDA lesion, SOD2 protein manifested obvious increase compared with control group (P < 0.01). Microinjection of DJ-1 protein was found to increase SOD2 protein expression compared with microinjection of 6-OHDA alone (P < 0.05).
The levels of ␣-synuclein and hypoxia-inducible factor 1␣ (HIF1␣) mRNA in the right SN of the MG-132 lesioned rats were detected using real-time PCR (Fig. 5A and B). Microinjection of MG-132 significantly increased ␣-synuclein and HIF-1␣ mRNA expression compared with control group (both P < 0.01). Administration of DJ1 protein was found to depress ␣-synuclein and HIF-1␣ mRNA expression apparently (both P < 0.01 vs. MG-132 group). Similar to mRNA results, ␣-synuclein protein expression in the right striatum of MG-132 group increased significantly compared with control group (P < 0.01), and DJ-1 protein obviously reduced ␣-synuclein protein expression in MG-132 + DJ-1 group (P < 0.01 vs. MG-132 group) (Fig. 5C and D). 4. Discussion In the present study, DJ-1 protein was microinjected into the MFB of PD rats those had been injected to 6-OHDA or MG-132.
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Fig. 5. DJ-1 protein inhibited ␣-synuclein expression and HIF-1␣ mRNA expression in the SN and striatum of dopaminergic neurons. (A and B) Levels of HIF-1␣ and ␣synuclein mRNA in the right SN of MG-132 lesioned rats detected by real-time PCR. (C and D) ␣-Synuclein protein expression in the right striatum of MG-132 lesioned rats detected by Western blot. Data are expressed as means ± SD or means (% of control) ± SD (n = 3). **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. MG-132 group.
Administration of DJ-1 protein apparently improved PD phenotypes of rats, including improving motor abnormality, inhibiting reduction of DA contents and TH levels in the striatum, and decreasing dopaminergic neuron death in the SN. Furthermore, by measuring a series of mRNA and protein in the SN and striatum, DJ-1 protein was found to protect dopaminergic neurons through diverse pathways. Traditional animal models of PD are based on the use of toxins, which selectively accumulate in the SN dopaminergic neurons, causing cellular dys-function and death, and 6-OHDA is the most frequently used one (Beal, 2010). In this experiment, results of 6OHDA lesiond rat model were consistent with previous reports (Baluchnejadmojarad et al., 2009; Inden et al., 2006). On the other hand, impairment of the ubiquitin–proteasome system (UPS) has been reported to play an important role in the pathogenesis of PD (Bedford et al., 2008). Microinjection with proteasome inhibitor MG-132 into the MFB or SN of C57BL/6 mice induced a sustained dopaminergic neurons degeneration, which recapitulated many neuropathological and behavioral features of PD (Sun et al., 2006; Xie et al., 2010). In our study, MG-132 was microinjected into the right MFB of rats, which induced movement disorders, DA and its metabolites contents and TH levels reduction in the striatum, and dopaminergic neuron degeneration in the SN. Microinjection of 6-OHDA into the MFB of rats leads to oxidative stimuli and generates ROS, which causes damage to mitochondria in dopaminergic neurons. UCPs are a class of mitochondrial ion channel, whose open probability is increased by superoxide (Krauss et al., 2005). Blocking UCPs may cause superoxide concentrations and oxidation of mitochondrial proteins to increase. Guzman et al. confirmed that knocking out DJ-1 downregulated the expression of UCPs (UCP4 and UCP5) and increased oxidation of matrix
proteins specifically in substantia nigra compacta (SNc) of dopaminergic neurons (Guzman et al., 2010). In our study, administration of DJ-1 protein increased the abundance of UCP4 and UCP5 mRNAs in the SN and thus might enhance mitochondrial uncoupling in response to oxidant stress, which made a significant contribution to maintain the stability of mitochondria and preserve dopaminergic neuron survival. In addition to UCPs, SOD2 also plays an important role when mitochondria suffers damage from oxidative stress (Guzman et al., 2010). SODs are a ubiquitous family of enzymes those function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian SODs have been biochemically and molecularly characterized. SOD2 is one of the three SODs located in the mitochondria and serves as the primary defense against mitochondrial superoxide (Lebovitz et al., 1996; Zelko et al., 2002). SOD2 mutation may increase PD risk especially for people who are frequently exposured to pesticides (Fong et al., 2007). The expression of human SOD2 could be induced by DJ-1 through increasing the activity of peroxisome proliferatoractivated receptor-␥ co-activator 1␣ (PGC-1␣) (Zhong and Xu, 2008). In our study, SOD2 mRNA and protein in the SN and striatum of PD model rats increased obviously after administration of DJ-1 protein into the MFB. When oxidative stress signals overwhelmed the ROS defense machinery after 6-OHDA administration, DJ-1 protein led to increased synergistic activation of the SOD2, thus slowing down the oxidative damage. mRNA and protein of SOD2 in rats of 6-OHDA group were also found to increase, which was consistent with previous reports (Carange et al., 2011; LahaieCollins et al., 2008). The reason for this phenomenon might be that SOD2 was increased by 6-OHDA induced superoxide ions under a response-to-stress mechanism. Therefore, we concluded that
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Hiroyoshi Ariga of Hokkaido University for providing the pGEX-6p1-GST DJ-1 plasmid.
References
Fig. 6. Proposed mechanisms by which DJ-1 protein protects against 6-OHDA and MG-132 toxicity.
DJ-1 protein increased antioxidant capacity to protect dopaminergic neurons from 6-OHDA damage. MG-132 is different with 6-OHDA in action mechanism, and it mainly results in irregular protein aggregation in the brain. Xie et al. certificated that microinjection with MG-132 into the MFB of mice led to sustained dopaminergic neuron degeneration with ␣-synuclein aggregation (Xie et al., 2010). ␣-Synuclein resided predominantly in the ‘presynaptic’ or output terminals of neurons, and stuck-together ␣-synuclein proteins constituted the tiny fibrils making up Lewy bodies, which were thought to be neuronal losses and pathological signs of PD (Schnabel, 2010). In our study, ␣-synulcein mRNA and protein were found to increase in MG-132 lesioned rats. Microinjection of DJ-1 protein obviously decreased ␣-synulcein at both protein and mRNA levels. Therefore, DJ-1 protein might protect dopaminergic neurons in the SN and striatum through reducing ␣-synulcein protein expression. Furthermore, we also found that HIF-1␣ mRNA in the dopaminergic neurons increased after administration of MG-132. HIF-1␣ is the only transcription factor associated with down-regulation of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrphic factor (GDNF), a neuroprotective molecule for dopaminergic neurons (Foti et al., 2010). HIF-1␣ overexpression may cause RET down-regulation and lead to loss of GDNF protection in dopaminergic neurons. Administration of DJ1 protein decreased HIF-1␣ mRNA expression and thus might repress down-regulation of RET, in which case ligand of RET, GDNF might promote survival of dopaminergic neurons in PD. Further study was needed to investigate molecular mechanism underlying this pathway. Therefore, we concluded that DJ-1 protein inhibited ␣-synulcein protein expression to protect dopaminergic neurons from MG-132 damage (Fig. 6). In summary, two toxins with different action mechanisms were used to modeling PD in rats. Simultaneous administration of DJ-1 protein into the MFB of rats dramatically improved PD phenotypes in two models. Besides, we also found that DJ-1 protein protected dopaminergic neurons through different pathways, including increasing antioxidant capacity and inhibiting ␣-synuclein expression. However, detailed molecular mechanisms were needed to be investigated in further study. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by the Doctor Fund of the Ministry of Education in China (20090001110069). We thank Professor
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