- Email: [email protected]
PAR2-mediated epigenetic upregulation of α-synuclein contributes to the pathogenesis of Parkinson׳s disease
brain research 1565 (2014) 82–89
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
PAR2-mediated epigenetic upregulation of α-synuclein contributes to the pathogenesis of Parkinson's disease Ping Liua,1, Liang Sunb,1, Xu-li Zhaoc, Peng Zhangd, Xue-mei Zhaoa, Jian Zhanga,n a
Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China c Department of Pain Management, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China d Department of Orthopedic, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China b
ar t ic l e in f o
abs tra ct
Article history:
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the
Accepted 11 April 2014
selective degeneration of projecting dopaminergic neurons in the substantia nigra and
Available online 18 April 2014
diminished dopamine levels in the striatum. Accumulating evidences demonstrate that
Keywords:
the aggregation of extracellular α-synuclein contributes to the neuroinflammation and
α-synuclein
neuronal injury in the substantia nigra in the brain of patients with PD. Proteinase-
Proteinase-activated receptor 2
activated receptor 2 (PAR2), a G-protein coupled receptor, is expressed throughout the
Nuclear factor-κB
peripheral and central nerve system. The present study aims to investigate the involve-
Parkinson's disease Histone acetylation
ment of PAR2–NF-κB signaling in the upregulation of α-synuclein and motor dysfunction in the rodent model of PD. Significantly increased expression of α-synuclein was observed in the substantia nigra of the rats injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In these rats, significantly increased nigral PAR2 was observed, and blockade of PAR2 signaling reduced the α-synuclein synthesis in substantia nigra and recovered the motor dysfunction in the rats injected with MPTP. Furthermore, significantly increased phosphorylation of NF-κB subunit p65 was detected in these rats, which was abolished by the inhibition of PAR2 signaling. Blockade of NF-κB signaling significantly decreased histone H3 acetylation in Snca promoter region and α-synuclein expression in substantia nigra. It also decreased the synthesis of cytokine IL-1β and TNF-α in substantia nigra and recovered the motor dysfunction in the rats injected with MPTP. These results indicated the critical involvement of PAR2–NF-κB signaling in the upregulation of α-synuclein and motor dysfunction in the rodent model of PD, and shed light on the development of novel approaches for the treatment of patients with PD. & 2014 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ86 531 68776465. E-mail address: [email protected] (J. Zhang). 1 These authors equally contributed to this work.
http://dx.doi.org/10.1016/j.brainres.2014.04.014 0006-8993/& 2014 Elsevier B.V. All rights reserved.
brain research 1565 (2014) 82–89
1.
Introduction
Parkinson's disease (PD) is a common neurodegenerative disorder with the symptoms including bradykinesia, rest tremor, rigidity, and postural and gait impairments (Massano and Bhatia, 2012). PD is characterized by the selective degeneration of projecting dopaminergic neurons in the substantia nigra and diminished dopamine levels in the striatum (Bagetta et al., 2010), and the presence of Lewy bodies and neurites containing α-synuclein aggregates in these central neurons (Dauer and Przedborski, 2003; Goedert, 2001). α-synuclein, encoding by Snca, generally binds synaptic vesicle membranes and potentially assists vesicle trafficking and SNARE complex formation (Norris et al., 2004; Rizo and Sudhof, 2012). Increase of α-synuclein in the presynaptic terminal was observed in the rodent model of PD (Spinelli et al., 2014), which substantially contributed to the microglia activation and neurodegeneration in the substantia nigra (Lee et al., 2014). Hence, understanding the molecular mechanism for the modulation of α-synuclein synthesis provided potential therapeutic opportunity for the patients with PD. Proteinase-activated receptors (PARs) are a family of G-protein coupled receptors that are activated by proteases, which liberates a tethered ligand, by cleaving the N-terminus of the receptors, and initiates several intracellular signal pathways (Rothmeier and Ruf, 2012). Four subtypes of PARs exist. While PAR1, PAR3, and PAR4 are preferentially activated by thrombin, PAR2 is preferentially activated by trypsin and trypsin-like proteinases (Hollenberg et al., 2014). All four PARs are expressed throughout the peripheral and central nerve system. While it was reported that the expression of PAR2 was significantly increased in the central nervous system in the rodent model of neruodegenerative diseases such as multiple sclerosis (Noorbakhsh et al., 2006) and Alzheimer's diseases (Afkhami-Goli et al., 2007), the involvement of PAR2 signaling in the pathogenesis of PD remained unexplored. While activation of G-protein couple receptor PAR2 generally triggers the mitogen-activated protein kinases signaling,
83
it also involves PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and induction of the Ca2þ/inositol 1,4,5-trisphosphate (IP3)/PKC signaling (Rothmeier and Ruf, 2012), which may in turn induce the phosphorylation of IKKα and IKKβ and result in the activation and nuclear translocation of NF-κB (Rothmeier and Ruf, 2012). Previous reports demonstrated that the increased NF-κB activity contributed to the neuronal injury in the rodent model of PD (Pranski et al., 2013; Williams et al., 2013). Phosphorylated NF-κB subunit p65, the active form of NF-κB, may bind to the promoter region, and increase the histone acetylation and facilitate the expression of target genes (Peng et al., 2011). Here, the present study aims to explore the potential involvement of PAR2–NF-κB signaling in the upregulation of α-synuclein in the rodent model of PD.
2.
Results
2.1. Increased α-synuclein in substantia nigra in the rats injected with MPTP As shown in Fig. 1a, injection of MPTP (25 mg/kg for 5 days) significantly decreased the time spent on the rod in the rats, which indicates an impaired motor balance. The ELISA study also found a significant increased expression of α-synuclein in the substantia nigra in these MPTP-injected rats (Fig. 1b), implying the potential role of α-synuclein in the pathogenesis of PD.
2.2. Increased PAR2 signaling in substantia nigra in the rats injected with MPTP Then we studied the expression and function of PAR2 signaling in the substantia nigra of the rats injected with MPTP. As shown in Fig. 2a, significantly increased expression of PAR2 was observed in the substantia nigra in the rats injected with MPTP, implying its potential role in the pathogenesis of PD in the rodent model.
Fig. 1 – Increased α-synuclein in the substantia nigra in rats injected with MPTP. (a) Significantly decreased time spent on the rotarod was observed in the rats injected with MPTP when compared with those injected with vehicle (n ¼11 and 13 rats in each group, Po0.01). (b) ELISA assay demonstrated an increased expression of α-synuclein in the substantia nigra in the rats injected with MPTP when compared with that in the vehicle-injected rats (n¼ 8 rats in each group, Po0.01). nn, Po0.01.
84
brain research 1565 (2014) 82–89
Fig. 2 – Increased PAR2 signaling in the substantia nigra in the rats-injected with MPTP. (a) Significantly increased expression in PAR2 in the substantia nigra tissue in the rats injected with MPTP when compared with that in the rats injected with vehicle (n ¼ 8 and 9 rats in each group, Po0.01). (b) Administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra attenuated the upregulation of α-synuclein expression in the substantia nigra in the MPTP-injected rats, while it did not change that in the vehicle-injected rats (n¼ 7–8 rats per group). (c) Administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra significantly extended the time spent in the rod in the MPTP-injected rats, while it did not change that in the vehicle-injected rats (n¼ 9–12 rats per group). n, Po0.05; nn, Po0.01.
To explore the functional significance of this upregulation of nigral PAR2 signaling, PAR2 antagonist FSLLRY-NH2 (2 μg) was daily delivered into the substantia nigra for 7 days. It was found that blockade of the PAR2 signaling significantly attenuated the expression of α-synuclein in the MPTP-injected rats, while it did not show an obvious effect in the control rats injected with vehicle (Fig. 2b). Further rotarod test showed a significantly increased time spent on the rod for the modeled rats after injected with PAR2 antagonist FSLLRY-NH2 (Fig. 2c). Note that inhibition of PAR2 signaling by FSLLRY-NH2 did not change the performance in the rotarod test in the control rats (Fig. 2c). These results indicated that upregulation of PAR2 signaling may modulate the expression of α-synuclein in substantia nigra, thus involved in the pathogenesis of PD in the rodent model.
2.3. Increased NF-κB phosphorylation and occupancy of NF-κB in α-synuclein promoter Activation of PAR2 signaling may induce the degradation of NF-κB inhibitor protein IkB, and lead to the activation and nuclear translocation of transcription factor NF-κB (Sriwai et al., 2013). Activation of NF-κB signaling is critically involved in the modulation of expression of target genes, thus participating in the induction and maintenance of central neuroinflammation. As shown in Fig. 3a, although the total expression level of NF-κB subunit p65 did not have significant change, the phosphorylated p65 was significantly increased in the substantia nigra of the rats injected with MPTP. It was further revealed that the increased
phosphorylated p65 was significantly recovered by the application of PAR2 antagonist FSLLRY-NH2 (2 μg for 7 days). Note that blockade of PAR2 signaling did not change the basal level of phosphorylated p65 in the control rats. These results suggested a PAR2 signaling-mediated upregulation of NF-κB signaling in the rats injected with MPTP. It was previously documented that phosphorylated NF-κB subunit p65 may bind to DNA sequence in the promoter region, and alter the histone acetylation in the promoter region and facilitate the expression of target genes. Here the ChIP study with polyclonal antibody against phosphorylated p65 subunit revealed a significant increased occupancy of p65 in the promoter region of Snca, but not Gapdh, in the substantia nigra of the rats injected with MPTP (Fig. 3b). Consistently, further ChIP studies also found a significantly increased acetylation of histone H3 in the promoter region of Snca, but not Gapdh, in the substantia nigra tissue of the MPTP-injected rats (Fig. 4a). A significantly increased mRNA of α-synuclein was also found in the AA of these modeled rats (Fig. 4b). These results indicated an NF-κB-mediated epigenetic modulation of α-synuclein in the substantia nigra of the rats injected with MPTP.
2.4. Blockade of NF-κB decreased α-synuclein acetyl-H3, mRNA and protein of α-synuclein and behavior To determine the functional significance of NF-κB-mediated epigenetic modulation of α-synuclein, pyrrolidine dithiocarbamate (PDTC, 2 μg for 7 days), an inhibitor of NF-κB, was
brain research 1565 (2014) 82–89
85
nigra of the modeled rats with the treatment of PDCT (Fig. 4c). Note that treatment with PDTC did not obviously change the histone H3 acetylation in the promoter region, mRNA and protein expression of α-synuclein in the control rats injected with vehicle (Fig. 4a–c). These results indicated that upregulated NF-κB signaling mediated the increased expression of α-synuclein in the substantia nigra in the rodent model of PD. Considering the role of α-synuclein to induce microglia activation and neuroinflammation, the further study also demonstrated that treatment with PDTC significantly attenuated the synthesis of cytokines IL-1β and TNF-α (Fig. 5a) in the substantia nigra. Moreover, treatment with PDTC significantly extended the time spent on the rod in the MPTP-injected rats, while it did not show a significant effect on the behavioral performance in the vehicle-injected rats (Fig. 5b). These results suggested that NF-κB-mediated epigenetic upregulation of α-synuclein might contribute to the neuroinflammation and motor dysfunction in the rodent model of PD.
3.
Fig. 3 – Increased NF-κB signaling in the substantia nigra in the rats injected with MPTP. (a) Significantly increased phosphorylation of NF-κB subunit p65, but not the total expression of p65, was observed in the substantia nigra in the MPTP-injected rats, which was substantially attenuated by the administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra (n ¼7–8 rats in each group). (b) Significantly increased binding of phosphorylated p65 was observed in the promoter region of Snca, but not Gapdh, in the MPTPinjected rats (n ¼6–7 rats per group). n, Po0.05; nn, Po0.01.
delivered into the substantia nigra in the rats injected with MPTP or vehicle. As shown in Fig. 4a, blockade of NF-κB signaling by PDTC significantly reversed the upregulation of histone H3 acetylation in the α-synuclein promoter region in the substantia nigra of the MPTP-injected rats. Further mRNA analysis revealed that PDTC significantly decreased the α-synuclein mRNA in the substantia nigra of the MPTPinjected rats (Fig. 4b). Consistently, a significant decreased expression of α-synuclein was also found in the substantia
Discussion
While it is predominantly expressed in neuronal synapses to regulate synaptic plasticity and neural differentiation under the physiological conditions, α-synuclein is also the major component of Lewy bodies in the neurological disorders including PD (Saracchi et al., 2014). It was previously reported that mutation of the gene encoding α-synuclein (Snca) was observed in the familial cases with early-onset PD (Polymeropoulos et al., 1997), and an accumulation of α-synuclein was substantially associated with the aggregate in oligomeric toxic compounds and the generation of the degenerative process of PD (Goedert and Spillantini, 1998). Remarkably elevated levels of oligomeric forms of α-synuclein were observed in the plasma samples obtained from clinical patients with PD (El-Agnaf et al., 2006). Aggregation of exogenous α-synuclein exhibited potent neurotoxicity to induce the neuronal apoptotic cell death and neurodegeneration (Luk et al., 2012). α-synuclein released from neuronal cells can be transferred to adjacent glia cells, and induce neuroinflammation characterized by the activation of microglia (Kim et al., 2013) and upregulation of cytokines (Reynolds et al., 2008). In the present study we also found a significantly increased expression of α-synuclein in the substantia nigra in the MPTP-injected rodent, and epigenetic suppression of α-synuclein was associated with the attenuation of cytokine upregulation and improvement of behavioral performance. These findings confirmed critical involvement of α-synuclein in the pathogenesis in the rodent model of PD. The present study further revealed a potential epigenetic mechanism, enhanced histone H3 acetylation in the promoter region, underlying the increased the α-synuclein synthesis in the substantia nigra in the MPTP-injected rodent. Usually acetylation of lysine residues reduces the positive charge on the histone N-terminal tails, and thus decreases the electrostatic attraction to the negatively charged backbone of DNA. This reduction in electrostatic attraction results in a looser chromatin structure allowing greater access of the transcriptional machinery to DNA, thereby facilitating the transcription of target gene (Rudenko and Tsai, 2014). Several types of histone acetyltransferases exist to catalyze the addition of acetyl groups on histone proteins (Maze et al., 2013). In the
86
brain research 1565 (2014) 82–89
Fig. 4 – Blockade of NF-κB decreased α-synuclein expression and behavioral impairment in the rats injected with MPTP. (a) MPTP significantly increased the histone H3 acetylation in the Snca, but not Gapdh, promoter region, which was recovered by administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra (n¼6–7 rats per group). (b) MPTP significantly increased the mRNA level of α-synuclein, which was recovered by administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra (n¼ 6–7 rats per group). (c) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly attenuated the upregulation of α-synuclein in the MPTP-injected rats (n¼ 6–7 rats per group). n, Po0.05; nn, Po0.01.
Fig. 5 – Blockade of NF-κB decreased cytokine synthesis and behavioral impairment in the rats injected with MPTP. (a) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly attenuated the upregulation of IL-1β and TNF-α in the MPTP-injected rats (n ¼6–7 rats per group). (b) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly extended the time spent on the rotarod in the MPTP-injected rats (n ¼ 10–12 rats per group). n , Po0.05; nn, Po0.01.
present study, an NF-κB-mediated histone acetyltransferase activity was revealed to participate in the epigenetic upregulation of α-synuclein in the substantia nigra of the rodent model of PD. Previous reports demonstrated the involvement of PAR2 signaling in the pathogenesis of several neurodegenerative
disorders (Afkhami-Goli et al., 2007; Noorbakhsh et al., 2006). The expression of PAR2 was significantly increased in the central nervous system in the rodent model of neruodegenerative diseases such as multiple sclerosis and Alzheimer's diseases, and modulation of PAR2 signaling largely influenced the development of these neurological disorders
brain research 1565 (2014) 82–89
(Afkhami-Goli et al., 2007; Noorbakhsh et al., 2006). In the present study, significantly increased expression of PAR2 was observed in the substantia nigra in the rodent model of PD, and inhibition of PAR2 signaling significantly attenuated the α-synuclein increase and motor dysfunction in the MPTPinjected rodent. These results indicated that the activation of PAR2 signaling played a pivotal role in the pathogenesis of PD in the rodent model. Upon activation, a PAR2 receptor may trigger several intracellular signal cascades involving MAPK signaling, PLC-mediated phospholipid signaling, and β-arrestin signaling (Rothmeier and Ruf, 2012). Among these, activation of PLC-mediated phospholipid signaling may induce the phosphorylation of IKKα and IKKβ and lead to activation and nuclear translocation of NF-κB (Rothmeier and Ruf, 2012; Sriwai et al., 2013). Phosphorylated subunit p65, the activated form of NF-κB, may bind to promoter region and increase the histone acetylation, thus facilitating the transcription and expression of target genes (Federman et al., 2013). A significantly increased NF-κB activity was previously reported in the substantia nigra of the rodent injected with MPTP (Roy et al., 2012), which contributed to the neuronal apoptotic death and dopamine deficits in the pathogenesis of PD (Mori et al., 2012; Pranski et al., 2013; Williams et al., 2013). In the present study, significantly increased phosphorylation of NF-κB subunit p65 was observed in the substantia nigra in the MPTPinjected rodent, and inhibition of NF-κB activity remarkably suppressed α-synuclein upregulation, cytokine synthesis, and motor imbalance in the rodent model of PD. These results indicated that NF-κB-mediated α-synuclein upregulation significantly contributed to the pathogenesis of PD in the present rodent model.
4.
Conclusion
The present study demonstrated that activation of PAR2 triggered NF-κB signaling and significantly upregulated the expression of α-synuclein via increasing the histone H3 acetylation in the promoter region of Snca, which substantially participated in the pathogenesis of PD. This indicated that PAR2–NF-κB signaling might become a novel target for the treatment of the clinical patients with PD.
5.
Experimental procedures
5.1. Animal model of Parkinson's disease and drug administration Adult male Wistar rats (200–220 g) were obtained from the Institutional Center of Experiment Animals, and were housed under the standard lab conditions (2272 1C and 12:12 h light cycle) with free access to food and water. All animal protocols were approved by the Institutional Animal Care and Use Committee, and were carried out following the guidelines of National Institution of Health. Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was performed as described previously (Yabuki et al., 2013). Rats were intraperitoneally treated with MPTP (25 mg/kg) or vehicle once a day for consecutive 5 days.
87
Rotarod test was performed at 14 days after the initial injection of MPTP to evaluate the motor balance and coordination in the animals.
5.2.
Cannula implantation and microinjection
The methods for site-specific cannula implantation and microinjection were similar as previously reported (Lessard and Couture, 2001). Before conditioning treatment, a rat was anesthetized with sodium pentobarbital solution (50 mg/kg). Animals were placed in a stereotaxic frame and were implanted with bilateral 26-gauge stainless steel chronic guide cannulae (Plastics One) into the substantia nigra (AP, 5.3; ML, 72.2; DV, 7.0) (Lessard and Couture, 2001). The guide cannula was then secured to skull with dental cement and capped. After the implantation surgery, the rat was allowed a recovery period for 1 week before subsequent experiments. Microinjection of FSLLRY-NH2 (2 μg), pyrrolidine dithiocarbamate (PDTC, 2 μg), or vehicle was performed daily for 7 days prior to the behavioral testing. The agents delivered into the substantia nigra through a 33-gauge single injector cannula with an infusion pump at a rate of 0.2 μl/min. 0.5 μl was injected on each side. All cannula placements for the substantia nigra were histologically verified afterwards.
5.3.
Motor performance testing
Motor performance was evaluated using a rotarod apparatus as previously described (Marques et al., 2014). The animals were placed in a rotarod with 60 mm diameter textured rod, 75 mm in length, rotating at a speed of 25 rpm. Each animal was tested 5 times with a 5 min interval between each trial and the maximum duration of the test was 5 min. The time spent by the animal on the rotarod was considered as the latency to fall.
5.4.
Protein extraction and immunoblotting
After behavioral testing, the rats were deeply anesthetized with pentobarbital sodium (50 mg/kg), and the substantia nigra tissues were quickly removed and homogenized in the lysis buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM NaVO3, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin with cocktails of phosphatase inhibitors ). The lysates were centrifuged at 14,000 rpm for 10 min at 4 1C, and the protein concentrations in the supernatant were measured by using the Bio-Rad protein assay kit. The samples (containing 15 μg proteins) were separated on a 7.5% SDSpolyacrylamide gel, and blotted to a nitrocellulose membrane. The blots were incubated overnight at 4 1C with a monoclonal anti-PAR2 antibody (1:1000; Millipore), polyclonal anti-NF-κB p65 (1:1000, Abcam), anti-phosphorylated NF-κB p65 (1:1000, Abcam), or monoclonal anti-β-actin antibody (1:1000; Santa Cruz Biotechnology). The membranes were washed extensively with Trisbuffered saline and incubated with horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG antibody (1:10,000; Jackson ImmunoResearch Laboratories). The immunoreactivity was detected using enhanced chemiluminescence. The intensity of the bands was captured digitally and analyzed quantitatively
88
brain research 1565 (2014) 82–89
with Image J software. The immunoreactivities of target proteins were normalized to those of β-actin.
5.5.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed with commercial ELISA kits to detect the content of α-synuclein (detect range: 1–500 ng/ml, Millipore), IL-1β and TNF-α (detect range: 1–1000 pg/ml, R & D Systems) in the substantia nigra as previously reported (Ma et al., 2014). The substantia nigra tissues from the rats in all groups were collected, and processed with commercial ELISA kits following the instructions provided by the manufacturer with minor modification.
5.6. PCR
Chromatin Immunoprecipitation (ChIP) and real-time
The ChIP assay was performed following the protocol provided by the manufacturer with minor modifications. The substantia nigra tissues from the rats were collected, and cross-linked with 1% formaldehyde for 5 min. Chromatin was solubilized in RIPA buffer (140 mM NaCl, 1 mM EDTA, 10 mM Tris–HCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycolate) and sonicated on ice 6 30 s to produce fragments of approximately 200–400 bp. One fifth of the lysate (40 μl) was saved to determine the input of DNA. The polyclonal antibodies against histone H3 acetylated at the lysine residues of N-terminus (K9, K14, K18 and K23) or antiphosphorylated p65 antibody (Millipore) were added and incubated overnight at 4 1C with gentle mixing. Immunocomplexes were recovered by the salmon sperm DNA/protein A agarose beads. The cross-link between histone and DNA was reversed, and DNA fragments were purified with phenol–chloroform extraction followed by acid ethanol precipitation. Real-time PCR was performed with the primers designed to amplify a 200-bp fragment ( from 422 to 223) in the promoter region of the Snca: 50 -GGCTGTGTGAACAAAAGCAA-30 and 50 -TGAACTTGAGCTGGCCTCTT-30 . The primers for Gapdh were used as following: 50 -AGACAGCCGCATCTTCTTGT-30 and 50 -CGTCCTCTACCATCCTCTGC-30 . The ChIP signal was analyzed as following: ΔCt[normalized ChIP] ¼ (Ct[ChIP] (Ct[Input] Log2(input dilution factor))); and ChIP/Input ratio¼ 2( ΔCt [normalized ChIP]).
5.7.
Retroscribed real-time PCR
Substantia nigra tissues were taken from the rats, and the total RNA was prepared with Trizol reagent (Invitrogen). Reverse transcribed PCR was performed by using a SYBR Green reverse transcription (RT)–PCR Reagents kit (Applied Biosystems, Foster City, CA). The primers were designed to amplify about 200 bP fragments within the cDNA sequence of the target genes as following: for α-synuclein: 50 -AGAAAACCAAGCAGGGTGTG -30 and 50 -GCTCCCTCCACTGTCTTCTG-30 ; and for GAPDH: 50 -AGACAGCCGCATCTTCTTGT-30 and 50 -CTTGCCGTGGGTAGAGTCAT-30 . Real-time quantitative PCR was performed using SYBR Green qPCR SuperMix (Invitrogen) and the ABI PRISM7300 Sequence Detection System. The β-actin gene was used as an internal normalizer. The threshold cycle (Ct) values for each sample were determined with the amplification plots within the logarithmic phase. Data were analyzed by using the 2 ΔΔCt method.
5.8.
Statistical analysis
All chemicals and reagents were purchased from SigmaAldrich (St. Louis, MO), unless specified otherwise. All data were presented as means7SEM, and statistically analyzed with student t test or ANOVA. The criterion for statistical significance was Po0.05.
Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (No. 81300964), the China Postdoctoral Science Foundation (No. 2013M531611) and the Scientific and Technological Programme for Traditional Chinese Medicine of Shandong Province (No. 2013-190).
r e f e r e n c e s
Afkhami-Goli, A., Noorbakhsh, F., Keller, A.J., Vergnolle, N., Westaway, D., Jhamandas, J.H., Andrade-Gordon, P., Hollenberg, M.D., Arab, H., Dyck, R.H., Power, C., 2007. Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer’s disease. J. Immunol. 179, 5493–5503. Bagetta, V., Ghiglieri, V., Sgobio, C., Calabresi, P., Picconi, B., 2010. Synaptic dysfunction in Parkinson’s disease. Biochem. Soc. Trans. 38, 493–497. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889–909. El-Agnaf, O.M., Salem, S.A., Paleologou, K.E., Curran, M.D., Gibson, M.J., Court, J.A., Schlossmacher, M.G., Allsop, D., 2006. Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J. 20, 419–425. Federman, N., de la Fuente, V., Zalcman, G., Corbi, N., Onori, A., Passananti, C., Romano, A., 2013. Nuclear factor kappaBdependent histone acetylation is specifically involved in persistent forms of memory. J. Neurosci. 33, 7603–7614. Goedert, M., Spillantini, M.G., 1998. Lewy body diseases and multiple system atrophy as alpha-synucleinopathies. Mol. Psychiatry 3, 462–465. Goedert, M., 2001. Alpha-synuclein and neurodegenerative diseases. Nat. Rev. Neurosci. 2, 492–501. Hollenberg, M.D., Mihara, K., Polley, D., Suen, J.Y., Han, A., Fairlie, D.P., Ramachandran, R., 2014. Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease. Br. J. Pharmacol 171, 1180–1194. Kim, C., Ho, D.H., Suk, J.E., You, S., Michael, S., Kang, J., Joong Lee, S., Masliah, E., Hwang, D., Lee, H.J., Lee, S.J., 2013. Neuronreleased oligomeric alpha-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat. Commun. 4, 1562. Lee, H.J., Bae, E.J., Lee, S.J., 2014. Extracellular alpha-synuclein-a novel and crucial factor in Lewy body diseases. Nat. Rev. Neurol. 10, 92–98. Lessard, A., Couture, R., 2001. Modulation of cardiac activity by tachykinins in the rat substantia nigra. Br. J. Pharmacol. 134, 1749–1759. Luk, K.C., Kehm, V., Carroll, J., Zhang, B., O’Brien, P., Trojanowski, J.Q., Lee, V.M., 2012. Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953. Ma, G.F., Chen, S., Yin, L., Gao, X.D., Yao, W.B., 2014. Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage
brain research 1565 (2014) 82–89
migration in vitro via the NF-kappaB pathway. Acta Pharmacol. Sin. 35, 195–202. Marques, M.R., Stigger, F., Segabinazi, E., Augustin, O.A., Barbosa, S., Piazza, F.V., Achaval, M., Marcuzzo, S., 2014. Beneficial effects of early environmental enrichment on motor development and spinal cord plasticity in a rat model of cerebral palsy. Behav. Brain Res. 263, 149–157. Massano, J., Bhatia, K.P., 2012. Clinical approach to Parkinson’s disease: features, diagnosis, and principles of management. Cold Spring Harb. Perspect. Med. 2, a008870. Maze, I., Noh, K.M., Allis, C.D., 2013. Histone regulation in the CNS: basic principles of epigenetic plasticity. Neuropsychopharmacology 38, 3–22. Mori, T., Hayashi, T., Su, T.P., 2012. Compromising sigma-1 receptors at the endoplasmic reticulum render cytotoxicity to physiologically relevant concentrations of dopamine in a nuclear factor-kappaB/Bcl-2-dependent mechanism: potential relevance to Parkinson’s disease. J. Pharmacol. Exp. Ther. 341, 663–671. Noorbakhsh, F., Tsutsui, S., Vergnolle, N., Boven, L.A., Shariat, N., Vodjgani, M., Warren, K.G., Andrade-Gordon, P., Hollenberg, M.D., Power, C., 2006. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Exp. Med. 203, 425–435. Norris, E.H., Giasson, B.I., Lee, V.M., 2004. Alpha-synuclein: normal function and role in neurodegenerative diseases. Curr. Top. Dev. Biol. 60, 17–54. Peng, C., Aron, L., Klein, R., Li, M., Wurst, W., Prakash, N., Le, W., 2011. Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons. J. Neurosci. 31, 12802–12815. Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E.S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W.G., Lazzarini, A.M., Duvoisin, R.C., Di Iorio, G., Golbe, L.I., Nussbaum, R.L., 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047. Pranski, E., Van Sanford, C.D., Dalal, N., Orr, A.L., Karmali, D., Cooper, D.S., Gearing, M., Lah, J.J., Levey, A.I., Betarbet, R., 2013.
89
NF-kappaB activity is inversely correlated to RNF11 expression in Parkinson’s disease. Neurosci. Lett. 547, 16–20. Reynolds, A.D., Glanzer, J.G., Kadiu, I., Ricardo-Dukelow, M., Chaudhuri, A., Ciborowski, P., Cerny, R., Gelman, B., Thomas, M.P., Mosley, R.L., Gendelman, H.E., 2008. Nitrated alpha-synuclein-activated microglial profiling for Parkinson’s disease. J. Neurochem. 104, 1504–1525. Rizo, J., Sudhof, T.C., 2012. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices—guilty as charged?. Annu. Rev. Cell Dev. Biol. 28, 279–308. Rothmeier, A.S., Ruf, W., 2012. Protease-activated receptor 2 signaling in inflammation. Semin. Immunopathol. 34, 133–149. Roy, A., Ghosh, A., Jana, A., Liu, X., Brahmachari, S., Gendelman, H.E., Pahan, K., 2012. Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson’s disease. PLoS One 7, e38113. Rudenko, A., Tsai, L.H., 2014. Epigenetic modifications in the nervous system and their impact upon cognitive impairments. Neuropharmacology 80C, 70–82. Saracchi, E., Fermi, S., Brighina, L., 2014. Emerging candidate biomarkers for Parkinson’s disease: a review. Aging Dis. 5, 27–34. Spinelli, K.J., Taylor, J.K., Osterberg, V.R., Churchill, M.J., Pollock, E., Moore, C., Meshul, C.K., Unni, V.K., 2014. Presynaptic alphasynuclein aggregation in a mouse model of Parkinson’s disease. J. Neurosci. 34, 2037–2050. Sriwai, W., Mahavadi, S., Al-Shboul, O., Grider, J.R., Murthy, K.S., 2013. Distinctive G protein-dependent signaling by proteaseactivated receptor 2 (PAR2) in smooth muscle: feedback inhibition of RhoA by cAMP-independent PKA. PLoS One 8, e66743. Williams, C.A., Lin, Y., Maynard, A., Cheng, S.Y., 2013. Involvement of NF Kappa B in potentiated effect of Mncontaining dithiocarbamates on MPP(þ) induced cell death. Cell Mol. Neurobiol. 33, 815–823. Yabuki, Y., Ohizumi, Y., Yokosuka, A., Mimaki, Y., Fukunaga, K., 2013. Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice. Neuroscience.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
PAR2-mediated epigenetic upregulation of α-synuclein contributes to the pathogenesis of Parkinson's disease Ping Liua,1, Liang Sunb,1, Xu-li Zhaoc, Peng Zhangd, Xue-mei Zhaoa, Jian Zhanga,n a
Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China c Department of Pain Management, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China d Department of Orthopedic, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China b
ar t ic l e in f o
abs tra ct
Article history:
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the
Accepted 11 April 2014
selective degeneration of projecting dopaminergic neurons in the substantia nigra and
Available online 18 April 2014
diminished dopamine levels in the striatum. Accumulating evidences demonstrate that
Keywords:
the aggregation of extracellular α-synuclein contributes to the neuroinflammation and
α-synuclein
neuronal injury in the substantia nigra in the brain of patients with PD. Proteinase-
Proteinase-activated receptor 2
activated receptor 2 (PAR2), a G-protein coupled receptor, is expressed throughout the
Nuclear factor-κB
peripheral and central nerve system. The present study aims to investigate the involve-
Parkinson's disease Histone acetylation
ment of PAR2–NF-κB signaling in the upregulation of α-synuclein and motor dysfunction in the rodent model of PD. Significantly increased expression of α-synuclein was observed in the substantia nigra of the rats injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In these rats, significantly increased nigral PAR2 was observed, and blockade of PAR2 signaling reduced the α-synuclein synthesis in substantia nigra and recovered the motor dysfunction in the rats injected with MPTP. Furthermore, significantly increased phosphorylation of NF-κB subunit p65 was detected in these rats, which was abolished by the inhibition of PAR2 signaling. Blockade of NF-κB signaling significantly decreased histone H3 acetylation in Snca promoter region and α-synuclein expression in substantia nigra. It also decreased the synthesis of cytokine IL-1β and TNF-α in substantia nigra and recovered the motor dysfunction in the rats injected with MPTP. These results indicated the critical involvement of PAR2–NF-κB signaling in the upregulation of α-synuclein and motor dysfunction in the rodent model of PD, and shed light on the development of novel approaches for the treatment of patients with PD. & 2014 Elsevier B.V. All rights reserved.
n
Corresponding author. Fax: þ86 531 68776465. E-mail address: [email protected] (J. Zhang). 1 These authors equally contributed to this work.
http://dx.doi.org/10.1016/j.brainres.2014.04.014 0006-8993/& 2014 Elsevier B.V. All rights reserved.
brain research 1565 (2014) 82–89
1.
Introduction
Parkinson's disease (PD) is a common neurodegenerative disorder with the symptoms including bradykinesia, rest tremor, rigidity, and postural and gait impairments (Massano and Bhatia, 2012). PD is characterized by the selective degeneration of projecting dopaminergic neurons in the substantia nigra and diminished dopamine levels in the striatum (Bagetta et al., 2010), and the presence of Lewy bodies and neurites containing α-synuclein aggregates in these central neurons (Dauer and Przedborski, 2003; Goedert, 2001). α-synuclein, encoding by Snca, generally binds synaptic vesicle membranes and potentially assists vesicle trafficking and SNARE complex formation (Norris et al., 2004; Rizo and Sudhof, 2012). Increase of α-synuclein in the presynaptic terminal was observed in the rodent model of PD (Spinelli et al., 2014), which substantially contributed to the microglia activation and neurodegeneration in the substantia nigra (Lee et al., 2014). Hence, understanding the molecular mechanism for the modulation of α-synuclein synthesis provided potential therapeutic opportunity for the patients with PD. Proteinase-activated receptors (PARs) are a family of G-protein coupled receptors that are activated by proteases, which liberates a tethered ligand, by cleaving the N-terminus of the receptors, and initiates several intracellular signal pathways (Rothmeier and Ruf, 2012). Four subtypes of PARs exist. While PAR1, PAR3, and PAR4 are preferentially activated by thrombin, PAR2 is preferentially activated by trypsin and trypsin-like proteinases (Hollenberg et al., 2014). All four PARs are expressed throughout the peripheral and central nerve system. While it was reported that the expression of PAR2 was significantly increased in the central nervous system in the rodent model of neruodegenerative diseases such as multiple sclerosis (Noorbakhsh et al., 2006) and Alzheimer's diseases (Afkhami-Goli et al., 2007), the involvement of PAR2 signaling in the pathogenesis of PD remained unexplored. While activation of G-protein couple receptor PAR2 generally triggers the mitogen-activated protein kinases signaling,
83
it also involves PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and induction of the Ca2þ/inositol 1,4,5-trisphosphate (IP3)/PKC signaling (Rothmeier and Ruf, 2012), which may in turn induce the phosphorylation of IKKα and IKKβ and result in the activation and nuclear translocation of NF-κB (Rothmeier and Ruf, 2012). Previous reports demonstrated that the increased NF-κB activity contributed to the neuronal injury in the rodent model of PD (Pranski et al., 2013; Williams et al., 2013). Phosphorylated NF-κB subunit p65, the active form of NF-κB, may bind to the promoter region, and increase the histone acetylation and facilitate the expression of target genes (Peng et al., 2011). Here, the present study aims to explore the potential involvement of PAR2–NF-κB signaling in the upregulation of α-synuclein in the rodent model of PD.
2.
Results
2.1. Increased α-synuclein in substantia nigra in the rats injected with MPTP As shown in Fig. 1a, injection of MPTP (25 mg/kg for 5 days) significantly decreased the time spent on the rod in the rats, which indicates an impaired motor balance. The ELISA study also found a significant increased expression of α-synuclein in the substantia nigra in these MPTP-injected rats (Fig. 1b), implying the potential role of α-synuclein in the pathogenesis of PD.
2.2. Increased PAR2 signaling in substantia nigra in the rats injected with MPTP Then we studied the expression and function of PAR2 signaling in the substantia nigra of the rats injected with MPTP. As shown in Fig. 2a, significantly increased expression of PAR2 was observed in the substantia nigra in the rats injected with MPTP, implying its potential role in the pathogenesis of PD in the rodent model.
Fig. 1 – Increased α-synuclein in the substantia nigra in rats injected with MPTP. (a) Significantly decreased time spent on the rotarod was observed in the rats injected with MPTP when compared with those injected with vehicle (n ¼11 and 13 rats in each group, Po0.01). (b) ELISA assay demonstrated an increased expression of α-synuclein in the substantia nigra in the rats injected with MPTP when compared with that in the vehicle-injected rats (n¼ 8 rats in each group, Po0.01). nn, Po0.01.
84
brain research 1565 (2014) 82–89
Fig. 2 – Increased PAR2 signaling in the substantia nigra in the rats-injected with MPTP. (a) Significantly increased expression in PAR2 in the substantia nigra tissue in the rats injected with MPTP when compared with that in the rats injected with vehicle (n ¼ 8 and 9 rats in each group, Po0.01). (b) Administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra attenuated the upregulation of α-synuclein expression in the substantia nigra in the MPTP-injected rats, while it did not change that in the vehicle-injected rats (n¼ 7–8 rats per group). (c) Administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra significantly extended the time spent in the rod in the MPTP-injected rats, while it did not change that in the vehicle-injected rats (n¼ 9–12 rats per group). n, Po0.05; nn, Po0.01.
To explore the functional significance of this upregulation of nigral PAR2 signaling, PAR2 antagonist FSLLRY-NH2 (2 μg) was daily delivered into the substantia nigra for 7 days. It was found that blockade of the PAR2 signaling significantly attenuated the expression of α-synuclein in the MPTP-injected rats, while it did not show an obvious effect in the control rats injected with vehicle (Fig. 2b). Further rotarod test showed a significantly increased time spent on the rod for the modeled rats after injected with PAR2 antagonist FSLLRY-NH2 (Fig. 2c). Note that inhibition of PAR2 signaling by FSLLRY-NH2 did not change the performance in the rotarod test in the control rats (Fig. 2c). These results indicated that upregulation of PAR2 signaling may modulate the expression of α-synuclein in substantia nigra, thus involved in the pathogenesis of PD in the rodent model.
2.3. Increased NF-κB phosphorylation and occupancy of NF-κB in α-synuclein promoter Activation of PAR2 signaling may induce the degradation of NF-κB inhibitor protein IkB, and lead to the activation and nuclear translocation of transcription factor NF-κB (Sriwai et al., 2013). Activation of NF-κB signaling is critically involved in the modulation of expression of target genes, thus participating in the induction and maintenance of central neuroinflammation. As shown in Fig. 3a, although the total expression level of NF-κB subunit p65 did not have significant change, the phosphorylated p65 was significantly increased in the substantia nigra of the rats injected with MPTP. It was further revealed that the increased
phosphorylated p65 was significantly recovered by the application of PAR2 antagonist FSLLRY-NH2 (2 μg for 7 days). Note that blockade of PAR2 signaling did not change the basal level of phosphorylated p65 in the control rats. These results suggested a PAR2 signaling-mediated upregulation of NF-κB signaling in the rats injected with MPTP. It was previously documented that phosphorylated NF-κB subunit p65 may bind to DNA sequence in the promoter region, and alter the histone acetylation in the promoter region and facilitate the expression of target genes. Here the ChIP study with polyclonal antibody against phosphorylated p65 subunit revealed a significant increased occupancy of p65 in the promoter region of Snca, but not Gapdh, in the substantia nigra of the rats injected with MPTP (Fig. 3b). Consistently, further ChIP studies also found a significantly increased acetylation of histone H3 in the promoter region of Snca, but not Gapdh, in the substantia nigra tissue of the MPTP-injected rats (Fig. 4a). A significantly increased mRNA of α-synuclein was also found in the AA of these modeled rats (Fig. 4b). These results indicated an NF-κB-mediated epigenetic modulation of α-synuclein in the substantia nigra of the rats injected with MPTP.
2.4. Blockade of NF-κB decreased α-synuclein acetyl-H3, mRNA and protein of α-synuclein and behavior To determine the functional significance of NF-κB-mediated epigenetic modulation of α-synuclein, pyrrolidine dithiocarbamate (PDTC, 2 μg for 7 days), an inhibitor of NF-κB, was
brain research 1565 (2014) 82–89
85
nigra of the modeled rats with the treatment of PDCT (Fig. 4c). Note that treatment with PDTC did not obviously change the histone H3 acetylation in the promoter region, mRNA and protein expression of α-synuclein in the control rats injected with vehicle (Fig. 4a–c). These results indicated that upregulated NF-κB signaling mediated the increased expression of α-synuclein in the substantia nigra in the rodent model of PD. Considering the role of α-synuclein to induce microglia activation and neuroinflammation, the further study also demonstrated that treatment with PDTC significantly attenuated the synthesis of cytokines IL-1β and TNF-α (Fig. 5a) in the substantia nigra. Moreover, treatment with PDTC significantly extended the time spent on the rod in the MPTP-injected rats, while it did not show a significant effect on the behavioral performance in the vehicle-injected rats (Fig. 5b). These results suggested that NF-κB-mediated epigenetic upregulation of α-synuclein might contribute to the neuroinflammation and motor dysfunction in the rodent model of PD.
3.
Fig. 3 – Increased NF-κB signaling in the substantia nigra in the rats injected with MPTP. (a) Significantly increased phosphorylation of NF-κB subunit p65, but not the total expression of p65, was observed in the substantia nigra in the MPTP-injected rats, which was substantially attenuated by the administration of FSLLRY-NH2 (2 μg for 7 days) into substantia nigra (n ¼7–8 rats in each group). (b) Significantly increased binding of phosphorylated p65 was observed in the promoter region of Snca, but not Gapdh, in the MPTPinjected rats (n ¼6–7 rats per group). n, Po0.05; nn, Po0.01.
delivered into the substantia nigra in the rats injected with MPTP or vehicle. As shown in Fig. 4a, blockade of NF-κB signaling by PDTC significantly reversed the upregulation of histone H3 acetylation in the α-synuclein promoter region in the substantia nigra of the MPTP-injected rats. Further mRNA analysis revealed that PDTC significantly decreased the α-synuclein mRNA in the substantia nigra of the MPTPinjected rats (Fig. 4b). Consistently, a significant decreased expression of α-synuclein was also found in the substantia
Discussion
While it is predominantly expressed in neuronal synapses to regulate synaptic plasticity and neural differentiation under the physiological conditions, α-synuclein is also the major component of Lewy bodies in the neurological disorders including PD (Saracchi et al., 2014). It was previously reported that mutation of the gene encoding α-synuclein (Snca) was observed in the familial cases with early-onset PD (Polymeropoulos et al., 1997), and an accumulation of α-synuclein was substantially associated with the aggregate in oligomeric toxic compounds and the generation of the degenerative process of PD (Goedert and Spillantini, 1998). Remarkably elevated levels of oligomeric forms of α-synuclein were observed in the plasma samples obtained from clinical patients with PD (El-Agnaf et al., 2006). Aggregation of exogenous α-synuclein exhibited potent neurotoxicity to induce the neuronal apoptotic cell death and neurodegeneration (Luk et al., 2012). α-synuclein released from neuronal cells can be transferred to adjacent glia cells, and induce neuroinflammation characterized by the activation of microglia (Kim et al., 2013) and upregulation of cytokines (Reynolds et al., 2008). In the present study we also found a significantly increased expression of α-synuclein in the substantia nigra in the MPTP-injected rodent, and epigenetic suppression of α-synuclein was associated with the attenuation of cytokine upregulation and improvement of behavioral performance. These findings confirmed critical involvement of α-synuclein in the pathogenesis in the rodent model of PD. The present study further revealed a potential epigenetic mechanism, enhanced histone H3 acetylation in the promoter region, underlying the increased the α-synuclein synthesis in the substantia nigra in the MPTP-injected rodent. Usually acetylation of lysine residues reduces the positive charge on the histone N-terminal tails, and thus decreases the electrostatic attraction to the negatively charged backbone of DNA. This reduction in electrostatic attraction results in a looser chromatin structure allowing greater access of the transcriptional machinery to DNA, thereby facilitating the transcription of target gene (Rudenko and Tsai, 2014). Several types of histone acetyltransferases exist to catalyze the addition of acetyl groups on histone proteins (Maze et al., 2013). In the
86
brain research 1565 (2014) 82–89
Fig. 4 – Blockade of NF-κB decreased α-synuclein expression and behavioral impairment in the rats injected with MPTP. (a) MPTP significantly increased the histone H3 acetylation in the Snca, but not Gapdh, promoter region, which was recovered by administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra (n¼6–7 rats per group). (b) MPTP significantly increased the mRNA level of α-synuclein, which was recovered by administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra (n¼ 6–7 rats per group). (c) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly attenuated the upregulation of α-synuclein in the MPTP-injected rats (n¼ 6–7 rats per group). n, Po0.05; nn, Po0.01.
Fig. 5 – Blockade of NF-κB decreased cytokine synthesis and behavioral impairment in the rats injected with MPTP. (a) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly attenuated the upregulation of IL-1β and TNF-α in the MPTP-injected rats (n ¼6–7 rats per group). (b) Administration of NF-κB inhibitor PDTC (2 μg for 7 days) into substantia nigra significantly extended the time spent on the rotarod in the MPTP-injected rats (n ¼ 10–12 rats per group). n , Po0.05; nn, Po0.01.
present study, an NF-κB-mediated histone acetyltransferase activity was revealed to participate in the epigenetic upregulation of α-synuclein in the substantia nigra of the rodent model of PD. Previous reports demonstrated the involvement of PAR2 signaling in the pathogenesis of several neurodegenerative
disorders (Afkhami-Goli et al., 2007; Noorbakhsh et al., 2006). The expression of PAR2 was significantly increased in the central nervous system in the rodent model of neruodegenerative diseases such as multiple sclerosis and Alzheimer's diseases, and modulation of PAR2 signaling largely influenced the development of these neurological disorders
brain research 1565 (2014) 82–89
(Afkhami-Goli et al., 2007; Noorbakhsh et al., 2006). In the present study, significantly increased expression of PAR2 was observed in the substantia nigra in the rodent model of PD, and inhibition of PAR2 signaling significantly attenuated the α-synuclein increase and motor dysfunction in the MPTPinjected rodent. These results indicated that the activation of PAR2 signaling played a pivotal role in the pathogenesis of PD in the rodent model. Upon activation, a PAR2 receptor may trigger several intracellular signal cascades involving MAPK signaling, PLC-mediated phospholipid signaling, and β-arrestin signaling (Rothmeier and Ruf, 2012). Among these, activation of PLC-mediated phospholipid signaling may induce the phosphorylation of IKKα and IKKβ and lead to activation and nuclear translocation of NF-κB (Rothmeier and Ruf, 2012; Sriwai et al., 2013). Phosphorylated subunit p65, the activated form of NF-κB, may bind to promoter region and increase the histone acetylation, thus facilitating the transcription and expression of target genes (Federman et al., 2013). A significantly increased NF-κB activity was previously reported in the substantia nigra of the rodent injected with MPTP (Roy et al., 2012), which contributed to the neuronal apoptotic death and dopamine deficits in the pathogenesis of PD (Mori et al., 2012; Pranski et al., 2013; Williams et al., 2013). In the present study, significantly increased phosphorylation of NF-κB subunit p65 was observed in the substantia nigra in the MPTPinjected rodent, and inhibition of NF-κB activity remarkably suppressed α-synuclein upregulation, cytokine synthesis, and motor imbalance in the rodent model of PD. These results indicated that NF-κB-mediated α-synuclein upregulation significantly contributed to the pathogenesis of PD in the present rodent model.
4.
Conclusion
The present study demonstrated that activation of PAR2 triggered NF-κB signaling and significantly upregulated the expression of α-synuclein via increasing the histone H3 acetylation in the promoter region of Snca, which substantially participated in the pathogenesis of PD. This indicated that PAR2–NF-κB signaling might become a novel target for the treatment of the clinical patients with PD.
5.
Experimental procedures
5.1. Animal model of Parkinson's disease and drug administration Adult male Wistar rats (200–220 g) were obtained from the Institutional Center of Experiment Animals, and were housed under the standard lab conditions (2272 1C and 12:12 h light cycle) with free access to food and water. All animal protocols were approved by the Institutional Animal Care and Use Committee, and were carried out following the guidelines of National Institution of Health. Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was performed as described previously (Yabuki et al., 2013). Rats were intraperitoneally treated with MPTP (25 mg/kg) or vehicle once a day for consecutive 5 days.
87
Rotarod test was performed at 14 days after the initial injection of MPTP to evaluate the motor balance and coordination in the animals.
5.2.
Cannula implantation and microinjection
The methods for site-specific cannula implantation and microinjection were similar as previously reported (Lessard and Couture, 2001). Before conditioning treatment, a rat was anesthetized with sodium pentobarbital solution (50 mg/kg). Animals were placed in a stereotaxic frame and were implanted with bilateral 26-gauge stainless steel chronic guide cannulae (Plastics One) into the substantia nigra (AP, 5.3; ML, 72.2; DV, 7.0) (Lessard and Couture, 2001). The guide cannula was then secured to skull with dental cement and capped. After the implantation surgery, the rat was allowed a recovery period for 1 week before subsequent experiments. Microinjection of FSLLRY-NH2 (2 μg), pyrrolidine dithiocarbamate (PDTC, 2 μg), or vehicle was performed daily for 7 days prior to the behavioral testing. The agents delivered into the substantia nigra through a 33-gauge single injector cannula with an infusion pump at a rate of 0.2 μl/min. 0.5 μl was injected on each side. All cannula placements for the substantia nigra were histologically verified afterwards.
5.3.
Motor performance testing
Motor performance was evaluated using a rotarod apparatus as previously described (Marques et al., 2014). The animals were placed in a rotarod with 60 mm diameter textured rod, 75 mm in length, rotating at a speed of 25 rpm. Each animal was tested 5 times with a 5 min interval between each trial and the maximum duration of the test was 5 min. The time spent by the animal on the rotarod was considered as the latency to fall.
5.4.
Protein extraction and immunoblotting
After behavioral testing, the rats were deeply anesthetized with pentobarbital sodium (50 mg/kg), and the substantia nigra tissues were quickly removed and homogenized in the lysis buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM NaVO3, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin with cocktails of phosphatase inhibitors ). The lysates were centrifuged at 14,000 rpm for 10 min at 4 1C, and the protein concentrations in the supernatant were measured by using the Bio-Rad protein assay kit. The samples (containing 15 μg proteins) were separated on a 7.5% SDSpolyacrylamide gel, and blotted to a nitrocellulose membrane. The blots were incubated overnight at 4 1C with a monoclonal anti-PAR2 antibody (1:1000; Millipore), polyclonal anti-NF-κB p65 (1:1000, Abcam), anti-phosphorylated NF-κB p65 (1:1000, Abcam), or monoclonal anti-β-actin antibody (1:1000; Santa Cruz Biotechnology). The membranes were washed extensively with Trisbuffered saline and incubated with horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG antibody (1:10,000; Jackson ImmunoResearch Laboratories). The immunoreactivity was detected using enhanced chemiluminescence. The intensity of the bands was captured digitally and analyzed quantitatively
88
brain research 1565 (2014) 82–89
with Image J software. The immunoreactivities of target proteins were normalized to those of β-actin.
5.5.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed with commercial ELISA kits to detect the content of α-synuclein (detect range: 1–500 ng/ml, Millipore), IL-1β and TNF-α (detect range: 1–1000 pg/ml, R & D Systems) in the substantia nigra as previously reported (Ma et al., 2014). The substantia nigra tissues from the rats in all groups were collected, and processed with commercial ELISA kits following the instructions provided by the manufacturer with minor modification.
5.6. PCR
Chromatin Immunoprecipitation (ChIP) and real-time
The ChIP assay was performed following the protocol provided by the manufacturer with minor modifications. The substantia nigra tissues from the rats were collected, and cross-linked with 1% formaldehyde for 5 min. Chromatin was solubilized in RIPA buffer (140 mM NaCl, 1 mM EDTA, 10 mM Tris–HCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycolate) and sonicated on ice 6 30 s to produce fragments of approximately 200–400 bp. One fifth of the lysate (40 μl) was saved to determine the input of DNA. The polyclonal antibodies against histone H3 acetylated at the lysine residues of N-terminus (K9, K14, K18 and K23) or antiphosphorylated p65 antibody (Millipore) were added and incubated overnight at 4 1C with gentle mixing. Immunocomplexes were recovered by the salmon sperm DNA/protein A agarose beads. The cross-link between histone and DNA was reversed, and DNA fragments were purified with phenol–chloroform extraction followed by acid ethanol precipitation. Real-time PCR was performed with the primers designed to amplify a 200-bp fragment ( from 422 to 223) in the promoter region of the Snca: 50 -GGCTGTGTGAACAAAAGCAA-30 and 50 -TGAACTTGAGCTGGCCTCTT-30 . The primers for Gapdh were used as following: 50 -AGACAGCCGCATCTTCTTGT-30 and 50 -CGTCCTCTACCATCCTCTGC-30 . The ChIP signal was analyzed as following: ΔCt[normalized ChIP] ¼ (Ct[ChIP] (Ct[Input] Log2(input dilution factor))); and ChIP/Input ratio¼ 2( ΔCt [normalized ChIP]).
5.7.
Retroscribed real-time PCR
Substantia nigra tissues were taken from the rats, and the total RNA was prepared with Trizol reagent (Invitrogen). Reverse transcribed PCR was performed by using a SYBR Green reverse transcription (RT)–PCR Reagents kit (Applied Biosystems, Foster City, CA). The primers were designed to amplify about 200 bP fragments within the cDNA sequence of the target genes as following: for α-synuclein: 50 -AGAAAACCAAGCAGGGTGTG -30 and 50 -GCTCCCTCCACTGTCTTCTG-30 ; and for GAPDH: 50 -AGACAGCCGCATCTTCTTGT-30 and 50 -CTTGCCGTGGGTAGAGTCAT-30 . Real-time quantitative PCR was performed using SYBR Green qPCR SuperMix (Invitrogen) and the ABI PRISM7300 Sequence Detection System. The β-actin gene was used as an internal normalizer. The threshold cycle (Ct) values for each sample were determined with the amplification plots within the logarithmic phase. Data were analyzed by using the 2 ΔΔCt method.
5.8.
Statistical analysis
All chemicals and reagents were purchased from SigmaAldrich (St. Louis, MO), unless specified otherwise. All data were presented as means7SEM, and statistically analyzed with student t test or ANOVA. The criterion for statistical significance was Po0.05.
Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (No. 81300964), the China Postdoctoral Science Foundation (No. 2013M531611) and the Scientific and Technological Programme for Traditional Chinese Medicine of Shandong Province (No. 2013-190).
r e f e r e n c e s
Afkhami-Goli, A., Noorbakhsh, F., Keller, A.J., Vergnolle, N., Westaway, D., Jhamandas, J.H., Andrade-Gordon, P., Hollenberg, M.D., Arab, H., Dyck, R.H., Power, C., 2007. Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer’s disease. J. Immunol. 179, 5493–5503. Bagetta, V., Ghiglieri, V., Sgobio, C., Calabresi, P., Picconi, B., 2010. Synaptic dysfunction in Parkinson’s disease. Biochem. Soc. Trans. 38, 493–497. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889–909. El-Agnaf, O.M., Salem, S.A., Paleologou, K.E., Curran, M.D., Gibson, M.J., Court, J.A., Schlossmacher, M.G., Allsop, D., 2006. Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J. 20, 419–425. Federman, N., de la Fuente, V., Zalcman, G., Corbi, N., Onori, A., Passananti, C., Romano, A., 2013. Nuclear factor kappaBdependent histone acetylation is specifically involved in persistent forms of memory. J. Neurosci. 33, 7603–7614. Goedert, M., Spillantini, M.G., 1998. Lewy body diseases and multiple system atrophy as alpha-synucleinopathies. Mol. Psychiatry 3, 462–465. Goedert, M., 2001. Alpha-synuclein and neurodegenerative diseases. Nat. Rev. Neurosci. 2, 492–501. Hollenberg, M.D., Mihara, K., Polley, D., Suen, J.Y., Han, A., Fairlie, D.P., Ramachandran, R., 2014. Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease. Br. J. Pharmacol 171, 1180–1194. Kim, C., Ho, D.H., Suk, J.E., You, S., Michael, S., Kang, J., Joong Lee, S., Masliah, E., Hwang, D., Lee, H.J., Lee, S.J., 2013. Neuronreleased oligomeric alpha-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat. Commun. 4, 1562. Lee, H.J., Bae, E.J., Lee, S.J., 2014. Extracellular alpha-synuclein-a novel and crucial factor in Lewy body diseases. Nat. Rev. Neurol. 10, 92–98. Lessard, A., Couture, R., 2001. Modulation of cardiac activity by tachykinins in the rat substantia nigra. Br. J. Pharmacol. 134, 1749–1759. Luk, K.C., Kehm, V., Carroll, J., Zhang, B., O’Brien, P., Trojanowski, J.Q., Lee, V.M., 2012. Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953. Ma, G.F., Chen, S., Yin, L., Gao, X.D., Yao, W.B., 2014. Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage
brain research 1565 (2014) 82–89
migration in vitro via the NF-kappaB pathway. Acta Pharmacol. Sin. 35, 195–202. Marques, M.R., Stigger, F., Segabinazi, E., Augustin, O.A., Barbosa, S., Piazza, F.V., Achaval, M., Marcuzzo, S., 2014. Beneficial effects of early environmental enrichment on motor development and spinal cord plasticity in a rat model of cerebral palsy. Behav. Brain Res. 263, 149–157. Massano, J., Bhatia, K.P., 2012. Clinical approach to Parkinson’s disease: features, diagnosis, and principles of management. Cold Spring Harb. Perspect. Med. 2, a008870. Maze, I., Noh, K.M., Allis, C.D., 2013. Histone regulation in the CNS: basic principles of epigenetic plasticity. Neuropsychopharmacology 38, 3–22. Mori, T., Hayashi, T., Su, T.P., 2012. Compromising sigma-1 receptors at the endoplasmic reticulum render cytotoxicity to physiologically relevant concentrations of dopamine in a nuclear factor-kappaB/Bcl-2-dependent mechanism: potential relevance to Parkinson’s disease. J. Pharmacol. Exp. Ther. 341, 663–671. Noorbakhsh, F., Tsutsui, S., Vergnolle, N., Boven, L.A., Shariat, N., Vodjgani, M., Warren, K.G., Andrade-Gordon, P., Hollenberg, M.D., Power, C., 2006. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Exp. Med. 203, 425–435. Norris, E.H., Giasson, B.I., Lee, V.M., 2004. Alpha-synuclein: normal function and role in neurodegenerative diseases. Curr. Top. Dev. Biol. 60, 17–54. Peng, C., Aron, L., Klein, R., Li, M., Wurst, W., Prakash, N., Le, W., 2011. Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons. J. Neurosci. 31, 12802–12815. Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E.S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W.G., Lazzarini, A.M., Duvoisin, R.C., Di Iorio, G., Golbe, L.I., Nussbaum, R.L., 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047. Pranski, E., Van Sanford, C.D., Dalal, N., Orr, A.L., Karmali, D., Cooper, D.S., Gearing, M., Lah, J.J., Levey, A.I., Betarbet, R., 2013.
89
NF-kappaB activity is inversely correlated to RNF11 expression in Parkinson’s disease. Neurosci. Lett. 547, 16–20. Reynolds, A.D., Glanzer, J.G., Kadiu, I., Ricardo-Dukelow, M., Chaudhuri, A., Ciborowski, P., Cerny, R., Gelman, B., Thomas, M.P., Mosley, R.L., Gendelman, H.E., 2008. Nitrated alpha-synuclein-activated microglial profiling for Parkinson’s disease. J. Neurochem. 104, 1504–1525. Rizo, J., Sudhof, T.C., 2012. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices—guilty as charged?. Annu. Rev. Cell Dev. Biol. 28, 279–308. Rothmeier, A.S., Ruf, W., 2012. Protease-activated receptor 2 signaling in inflammation. Semin. Immunopathol. 34, 133–149. Roy, A., Ghosh, A., Jana, A., Liu, X., Brahmachari, S., Gendelman, H.E., Pahan, K., 2012. Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson’s disease. PLoS One 7, e38113. Rudenko, A., Tsai, L.H., 2014. Epigenetic modifications in the nervous system and their impact upon cognitive impairments. Neuropharmacology 80C, 70–82. Saracchi, E., Fermi, S., Brighina, L., 2014. Emerging candidate biomarkers for Parkinson’s disease: a review. Aging Dis. 5, 27–34. Spinelli, K.J., Taylor, J.K., Osterberg, V.R., Churchill, M.J., Pollock, E., Moore, C., Meshul, C.K., Unni, V.K., 2014. Presynaptic alphasynuclein aggregation in a mouse model of Parkinson’s disease. J. Neurosci. 34, 2037–2050. Sriwai, W., Mahavadi, S., Al-Shboul, O., Grider, J.R., Murthy, K.S., 2013. Distinctive G protein-dependent signaling by proteaseactivated receptor 2 (PAR2) in smooth muscle: feedback inhibition of RhoA by cAMP-independent PKA. PLoS One 8, e66743. Williams, C.A., Lin, Y., Maynard, A., Cheng, S.Y., 2013. Involvement of NF Kappa B in potentiated effect of Mncontaining dithiocarbamates on MPP(þ) induced cell death. Cell Mol. Neurobiol. 33, 815–823. Yabuki, Y., Ohizumi, Y., Yokosuka, A., Mimaki, Y., Fukunaga, K., 2013. Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice. Neuroscience.