J. Neurochem. (2012) 123, 417–427.
Neurokinin 3 (NK3) receptor is predominantly expressed in striatum and substantia nigra (SN). Evidences have indicated the roles of NK3 receptor in the pathogenesis of Parkinson’s disease. By administrating NK3 receptor agonist senktide into 6-hydroxydopamine (6-OHDA)-lesioned rats, exacerbation of dopaminergic degeneration was found in striatum and substantia nigra pars compacta. From apomorphine rotation test, significant increase of contralateral rotation number was detected in 6-OHDA-lesioned rats with senktide injection. Furthermore, tyrosine hydroxylase expression in striatum and substantia nigra pars compacta were examined by immunohistochemistry and Western blotting. Further reduction of tyrosine hydroxylase immunoreactivities was found in 6-OHDA-lesioned rats that received senktide treatment. Also, phosphorylation of N-methyl-D-aspartate receptor 1 subunit was investigated in SN region and significant up-regulation was revealed in senktide-treated 6-OHDA-lesioned rats. Finally, phosphorylation of mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) and c-Jun were examined in nigral region. Up-regulation of phosphorylated JNK molecules was shown in SN region after senktide injection. In line with this evidence, phosphorylation of c-Jun at Ser 63 and Ser 73 was also up-regulated by senktide treatment, thus presenting new aspects that NK3 peptide could exacerbate 6-OHDA toxicity in in vivo models and the possible mechanism may be contributed by the modulation of N-methyl-D-aspartate receptor 1 subunit and JNK pathway activities.
c Jun N-terminal kinase
- NR 1
N-methyl-D-aspartate receptor 1 subunit
phosphate buffer saline
substantia nigra pars compacta
Parkinson’s disease (PD) is a common neurodegenerative disease resulting from the degeneration of dopaminergic neurons in substantia nigra pars compacta (SNc) with the symptoms including resting tremor, slowness of movement, and rigidity of muscles. Even though various pathogenic factors were raised over the past few decades, the critical mechanism still remained largely unknown. Recently, mammalian neurokinin was suggested to be involved in the onset of PD (Barker 1996; Severini et al. 2002; Chen et al. 2004). Regulations of neurokinin have presented as potential candidates for pharmacological treatment in PD (Severini et al. 2002; Chen et al. 2004, 2008; Salthun-Lassalle et al. 2005).
Neurokinins are a group of neuropeptides that are widely distributed within different regions of CNS (Otsuka and Yoshioka 1993; Maggi 2000; Chen et al. 2004). There are three members of neurokinin: substance P (SP), neurokinin A, and neurokinin B, in which their physiological activities are monitored by NK1, NK2, and NK3 receptors, respectively (Nakanishi 1991; Maggi et al. 1993; Khawaja and Rogers 1996). The natural neurokinin 1 peptide SP is the most studied neurokinin member. Numerous reports have demonstrated the roles of SP and NK1 receptor in PD, including the neuroprotective effects of NK1 peptide in cell death models (Calvo et al. 1996; Lallemend et al. 2003; Amadoro et al. 2007; Chu et al. 2011).
On the other hand, the roles of NK3 peptide in PD progression are not fully understood yet. Meanwhile, from previous reports, localization of NK3 receptor was found in both striatum and substantia nigra (SN), which are the major dopaminergic degeneration sites in PD (Dam et al. 1990; Ding et al. 1996; Chen et al. 1998; Langlois et al. 2001). Also, NK3 receptors were shown to be involved in regulating dopaminergic transmission, thus indicating indirect evidences for the involvement of NK3 receptor in PD (Bannon et al. 1995; Alonso et al. 1996; Marco et al. 1998). Recently, NK3 receptors were also demonstrated to regulate glutamate-driven N-methyl-D-aspartate (NMDA) receptor activation and subsequent excitotoxicity (Severini et al. 2003; Wang et al. 2005; Chen et al. 2008). It has been shown that glutamate-induced excitotoxicity could result in intracellular calcium influx and initiates cell death (Nakanishi 1992; Doble 1999; Arundine and Tymianski 2003). One of the stress-stimulated intracellular pathways: the c Jun N-terminal kinase (JNK) pathway could be activated by calcium influx (Yang et al. 1997; Centeno et al. 2007). JNK pathway is a MAP kinase pathway, which is responsible for stress stimulus and cell death (Davis 1994; Ichijo 1999; Leppa and Bohmann 1999; Kyriakis and Avruch 2001; Brecht et al. 2005). Previous researchers have discovered the relationship between activation of JNK pathway and onset of neuronal degeneration (Peng and Andersen 2003). In view of these results, we hypothesized that NK3 peptide, instead of NK1 peptide, may contribute to the neuronal degeneration by modulating activating status of N-methyl-D-aspartate receptor 1 subunit and JNK pathway. NK3 peptide may over-activate NMDA receptor and this in turn leads to overloading of intracellular calcium in dopaminergic neurons. All these events would finally result in activating JNK pathway and cell death.
This study revealed the effects of a synthetic NK3 peptide, senktide, in 6-hydroxydopamine (6-OHDA)-lesioned rat model. Throughout the experiment, administration of senktide could deteriorate dopaminergic degeneration in 6-OHDA-lesioned rats. Besides, up-regulations of phosphorylated NR1, JNK and c-Jun molecules have been found in SN after senktide injection. Effects of senktide could be counteracted by co-administering NK3 receptor antagonist SB218795. To conclude, our results have presented novel data on the potential roles of NK3 peptide and NK3 receptor in in vivo 6-OHDA-lesioned model.
Materials and methods
Animals and tissue preparation
Adult male Sprague–Dawley rats weighing 150 g–200 g were obtained from University of Hong Kong. The handling of rats and all procedures were in accordance with the Animals (Control of Experiments) Ordinance, Hong Kong, China, and approved by the Committee on the Use of Human and Animal Subjects in Teaching and Research, Hong Kong Baptist University, and conformed to The Principles of Laboratory Animal Care (NIH publication No. 86–23, revised 1985).
Striatal injection of 6-OHDA and senktide
Rats were anesthetized with sodium pentobarbital (20 mg/kg, i.p., Nembutal, Abbott Laboratories) and placed in a stereotaxic apparatus. Two small holes were drilled on the skull after incision of the scalp. Saline or 6-OHDA injections were carried out into the right striatum as indicated in the following stereotaxic coordinates: site 1 injection: Bregma −2.6 mm rostracaudally, +1.4 mm laterally, and 5.00 mm depth from dura mater, site 2 injection: Bregma −3.8 mm rostracaudally, −0.4mm laterally, and 5.00 mm depth from dura mater according to the rat atlas of Paxinos and Watson (Paxinos and Watson 1986). 6-OHDA solution was freshly prepared by dissolving 6-OHDA (0.2 μg/μL, Sigma, St. Louis, MO, USA) in 0.9% saline with ascorbic acid. Two doses of 6 μL 6-OHDA solution were injected into right striatum according to previous reports (Cadet et al. 1989; Sauer et al. 1995). Same amount of 0.9% saline was injected into other groups of rats as control groups. After 7-days 6-OHDA treatment, second injection was carried out in 6-OHDA-lesioned rats with 7 μL of 0.9% saline, senktide solution (0.04 μg, Calbiochem, Billerica, MA, USA) or senktide with NK3 receptor antagonist SB218795 (0.1 μg, Sigma) at site 1 of striatum. The concentration of senktide and SB218795 was adopted with reference to the effective dosage used in previous experiments (Chen et al. 2008). On the other hand, same amount of 0.9% saline or same dosage of senktide solution was injected into control animals as vehicle control (sham operation) and senktide control (senktide-alone) groups. This protocol was adopted to ensure the efficiency of 6-OHDA lesion surgery prior to NK3 peptide or NK3 antagonist treatment (Table 1).
|Groups of animals||Reagents given in each group|
|Vehicle control (sham operation)||12μL saline (1st) + 7μL saline (2nd)|
|Senktide control (senktide alone)||12μL saline (1st) + 7μL senktide (2nd)|
|6-OHDA lesion||12μL 6-OHDA (1st) + 7μL saline (2nd)|
|6-OHDA + Senktide||12μL 6-OHDA (1st) + 7μL senktide (2nd)|
|6-OHDA + Senktide + SB218795||12μL 6-OHDA (1st) + 7μL senktide with SB218795 (2nd)|
Apomorphine rotation test
Throughout the experiment, animals were subjected to apomorphine (0.5 mg/kg, s.c., Sigma) on the day before senktide injection (6 days post-6-OHDA injection), 2 days after senktide injection (9 days post-6-OHDA injection), and 7 days after senktide injection (14 days post-6-OHDA injection). Concentration of apomorphine was adopted according to previous reports (Cadet et al. 1989; Przedborski et al. 1995). Counting of rotation number was started after 3-min apomorphine administration. The number of anticlockwise rotations was counted for 60 min and recorded for analysis.
Perfusion and tissue preparation
After treatments, rats were deeply anesthetized with sodium pentobarbital (60mg/kg, i.p., Nembutal, Abbott Laboratories, North Chicago, IL, USA). They were perfused with 300 mL 0.9% saline and then 300mL of 3% paraformaldehyde (Sigma) with 0.12 mL of 0.1% glutaldehyde (Sigma) in phosphate buffer (pH 7.4). The brain was dissected out and placed in 3% paraformaldehyde overnight at 4°C. Afterward, brain samples containing striatum and SN were serially cut into coronal 70-μm thickness sections on vibrotome and collected in phosphate buffer saline (PBS) (pH 7.4) for immunohistochemical staining.
Brain sections were first incubated with anti-TH antibody (1 : 2000 dilution; Millipore) in PBS containing 0.2% triton X-100 (USB, Cleveland, OH, USA) and 2% normal goat serum (Vector Labs, Burlingame, CA, USA) overnight, then washed with PBS three times and incubated with Alexa Fluo 488-conjugated antibody (1 : 500 dilution; Invitrogen, Carlsbad, CA, USA). These brain sections were examined under a confocal laser microscope and images were captured. TH-immunoreactivity was revealed in striatum and TH neurons in SNc were counted to quantify dopaminergic degeneration.
Rats were decapitated after 2-days or 7-days NK3 peptide treatment. Fresh nigral and striatal tissues were dissected out and stored in protein extraction buffer (Novagen, Billerica, MA, USA) supplemented with protease inhibitor (Calbiochem). Tissues were homogenized and centrifuged at 14 000 g. The protein supernatant was extracted and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Separated proteins were then transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) and incubated with different primary antibodies followed by respective horseradish peroxidase -conjugated secondary antibodies (Sigma). Anti- TH was from Millipore (Billerica, MA, USA). Anti- ß-actin was from sigma. Anti- phosphorylated JNK, anti- JNK, anti- phosphorylated c-Jun (Ser 63), anti- phosphorylated c-Jun (Ser 73), anti- c-Jun, and anti- phosphorylated NR1 were from Cell Signaling (Beverly, MA, USA). Protein bandings were visualized using enhanced chemiluminescence reagents (Ab Frontier, Seoul, Korea). Semiquantifications of the signals were performed using densitometry analysis of the Metamorph software.
Comparisons between different groups of treatment were analyzed by paired t-tests, using pasw Statistics 18 software. Significant differences were considered when p ≤ 0.05 or lower.
Apomorphine-induced contralateral rotation number was increased by senktide treatment in 6-OHDA-lesioned rats
Upon 6-OHDA treatment, degeneration of dopaminergic neurons and terminals occurred in SNc and striatum, resulting in deficiency of dopamine in striatum. The non-dopaminergic dopamine receptor agonist apomorphine was subcutaneously administrated with reference to previous experiments (Kirik et al. 1998; Bove et al. 2005). In 6-OHDA-treated groups, after 2-days senktide injection, no significant increase of rotation number was observed (Fig. 1). However, after 7-days senktide treatment, significant increase in rotation number was detected in senktide-treated 6-OHDA group compared with 6-OHDA-alone group. Simultaneously, the effect could be restored by SB218795 coadministration (Fig. 1). Throughout the experiment, no motor deficit was observed in both vehicle and senktide control groups (data not shown), thus demonstrating that no significant dopaminergic degeneration occurred in these two groups.
Loss of TH expression in striatum and SNc by 6-OHDA were exacerbated by senktide treatment
TH immunohistochemistry was carried out to visualize dopaminergic terminals and neurons in striatum and SNc. Loss of TH expression was also quantified by Western blotting. In 6-OHDA-lesioned rats, loss of TH immunoreactivity was found in SNc and striatum, respectively (Fig. 2). However, no significant exacerbation of neurodegeneration was found in 6-OHDA-lesioned rats with 2-days senktide treatment. Simultaneously, a similar trend was shown by Western blotting (Fig. 2). No significant exacerbation of dopaminergic degeneration was observed among 6-OHDA-lesioned groups. In contrast, deterioration was found in 6-OHDA-lesioned rats with 7-days senktide treatment. Significant loss of TH-positive neurons and terminal were found in comparison with 6-OHDA-alone group (Fig. 3). Western blotting also revealed a higher percentage loss of TH expression in 6-OHDA-lesioned rats after 7-days senktide treatment (Fig. 3). This effect could be reversed by coadministration of NK3 receptor antagonist SB218795. In vehicle and senktide control group, no remarkable TH loss was detected in striatum and SNc, implying that surgery and senktide itself did not cause significant damage to dopaminergic system (Fig. 2 and 3).
Phosphorylation of NR1 was up-regulated in SN of senktide-treated 6-OHDA-lesioned rats
Phosphorylation of NR subunit is one of the parameters reflecting the activity of NMDA receptor (Nakanishi 1992). Phosphorylation of NR1 subunit in SN region was compared by Western blotting. With 2-days senktide treatment, all groups of rats with senktide treatment demonstrated up-regulation of phosphorylated NR1 expression in SN region (Fig. 4). With 7-days senktide treatment, up-regulation of phosphorylated NR1 in SN region was still observed in senktide control group and senktide plus 6-OHDA-lesioned group, while SB218795 could suppress the up-regulation of NR1 phosphorylation in senktide-treated 6-OHDA-lesioned animals (Fig. 4), suggesting NK3 peptide was involved in regulating NMDA receptor activation.
NK3 agonist activated stress-activated protein kinase/JNK pathway and its downstream effector c-Jun in 6-OHDA-lesioned rats
The intervention mechanism of NK3 peptide on deteriorating 6-OHDA-lesioned process was further studied by examining the expression of phosphorylated JNK and c-Jun molecules in nigral region. Significant increase of phosphorylated JNK and c-Jun expression in SN was observed in 6-OHDA-lesioned rats with 2-days senktide treatment (Fig. 5 and 6). With 7-days senktide and Sb218795 treatment, up-regulations of phosphorylated JNK and c-Jun in senktide-treated groups were detected, while this up-regulation was counteracted by SB218795 coadministration (Fig. 5 and 6). These results implied that JNK pathway was involved in the senktide-induced exacerbation of 6-OHDA dopaminergic degeneration.
NK3 receptors are abundantly distributed within CNS, which was demonstrated by radioligand binding assays in previous reports (Ding et al. 1996; Ding et al. 1996; Langlois et al. 2001). Senktide is a specific NK3 receptor agonist that it demonstrated high affinity towards NK3 receptors and elicited physiological responses in cells through NK3 receptor (Keegan et al. 1992; Chen et al. 2008; Nordquist et al. 2008). Here, we reported that senktide could synergistically deteriorate dopaminergic degeneration in 6-OHDA-lesioned rat and the possible mechanism could be accounted by the modulation of NR1 phosphorylation and activation of JNK pathway. Taking together with our previous reports, NK3 peptide may involve in the process of neurodegeneration in in vivo model by regulating NR1 subunit and JNK pathway, thus promoting the onset of PD progress (Chen et al. 1998, 2008; Wang et al. 2005).
According to previous reports, complete degeneration of dopaminergic neurons could be observed in 6-OHDA-lesioned rats with 4–6 weeks of 6-OHDA injection (Beal 2001). Upon 6-OHDA treatment, progressive dopaminergic degeneration in SNc occurred at the first week and it was recognized as the early event of this PD model (Berger et al. 1991; Beal 2001). As deterioration of degeneration may not be able to compare between animal groups if complete lesioned achieved, early stage of 6-OHDA-lesioned models was chosen to access the severity of PD symptoms in 6-OHDA-lesioned animals after NK3 peptide and NK3 antagonist treatment.
As described before, neurokinin peptides are actively involved in regulating neuronal activities or even neuronal survival (Otsuka and Yoshioka 1993; Chen et al. 2004; Salthun-Lassalle et al. 2005). First, different studies have demonstrated the correlation between SP and the onset of PD (Tenovuo et al. 1990; Severini et al. 2002; Chen et al. 2004). Neuroprotective effects of NK1 peptide were described in various experimental models (Calvo et al. 1996; Lallemend et al. 2003; Amadoro et al. 2007; Chu et al. 2011). On the other hand, NK3 receptor was shown to be colocalized with cholinergic and dopaminergic neurons (Chen et al. 1998; Marco et al. 1998). Activation of NK3 receptor could also modulate dopaminergic neurons’ activities (Bannon et al. 1995; Alonso et al. 1996; Zhang et al. 2008). However, the critical linkages between activation of NK3 receptor and neurodegeneration are still under investigation. This study, in combination with our previous report, has illustrated that administration of NK3 peptide could enhance neurotoxin toxicity in dopaminergic neurons (Chen et al. 2008). And this effect was contributed by phosphorylation of NR1 and activation of JNK pathway.
From present results, deterioration of motor deficit was not shown in 6-OHDA-lesioned rats with 2-days senktide injection. However, significant increase of rotation number was found in 6-OHDA-lesioned rats when treatment time was extended to 7 days compared with 6-OHDA-alone group. Besides, no behavior deficit was seen in vehicle and senktide control group. This observation indicated that NK3 peptide induced further motor deficit in 6-OHDA-lesioned rats. As colocalization of NK3 receptor was previously shown in dopaminergic neurons (Bannon et al. 1995; Alonso et al. 1996; Chen et al. 1998), we provided direct evidences that NK3 peptide could accelerate dopamine loss in in vivo model upon insulting with neurotoxin or cellular stress, which was reflected by the increase of rotation number in apomorphine test.
Previously, phosphorylation of NR subunit was described as one of the parameters reflecting the activity of NMDA receptor (Nakanishi 1992), in which this event is closely related to excitotoxicity occurred in neurons (Beal 1992; Blandini et al. 1996, 2000). In companion with extra calcium influx, calcium-dependent protein kinase would be activated (Blandini et al. 2000; Mattson 2007). From previous cases, antagonizing of NMDA receptor has been shown to improve PD conditions. For instance, competitive NMDA antagonist could ameliorate parkinsonian symptoms in animal models of PD (Loftis and Janowsky 2003). Also, specific NMDA receptor antagonist was examined and showed anti-parkinsonian activities (Karcz-Kubicha et al. 1999; Loftis and Janowsky 2003). In present observations, significant up-regulation of phosphorylated-NR1 appeared in animals with 2- and 7-days senktide treatment, implying that NK3 peptide may trigger NR1 activation in dopaminergic neurons. As NR 1 and NK3 receptor were shown to be colocalized with dopaminergic neurons (Wang et al. 2005), dopaminergic neurons maybe vulnerable to excess calcium influx and activation of NK3 receptor would accelerate the degeneration through NMDA receptor-dependent excitotoxicity.
Furthermore, throughout the experiment, up-regulation of phosphorylated JNK and c-Jun molecules were demonstrated in SN region of senktide-treated 6-OHDA-lesioned rats. JNK is one of the members of mitogen-activated protein kinase. Classically, phosphorylation of JNK molecule could regulate gene transcription by c-Jun phosphorylation (Davis 1994; Leppa and Bohmann 1999; Kyriakis and Avruch 2001), in which both processes could mediate cell death (Leppa and Bohmann 1999; Brecht et al. 2005; Shen and Liu 2006; Johnson and Nakamura 2007). Recent evidences have also proposed that excitotoxicity or intracellular calcium influx could lead to the activation of calcium-dependent kinases and subsequent activation of JNK and c-Jun (Enslen et al. 1996; Yang et al. 1997; Kim and Sharma 2004; Olofsson et al. 2008). c-Jun could then cooperate with other transcriptional factors such as AP-1 or c-Fos (Karin et al. 1997; Shaulian and Karin 2002) and resulted in cell death (Peng and Andersen 2003; Borsello and Forloni 2007).
In line with these results, we have identified both up-regulation of phosphorylated NR1, JNK, and c-Jun in senktide-treated 6-OHDA-lesioned rats. On the basis of these findings, we suggested that NK3 peptide may play a crucial role in neuronal degeneration through modulation of NR1 and JNK pathway. With senktide injection, activated NK3 receptor triggered calcium influx (by NR1 phosphorylation) and catalyzed cell death process in 6-OHDA-lesioned rats by up-regulating intracellular JNK and c-Jun activities through calcium-dependent kinases. Finally, the deterioration of dopaminergic degeneration was reflected by the motor deficit in apomorphine rotation test. However, one interesting observation was that throughout the experiment, both changes in NR1 phosphorylation and JNK pathway were found in both 2-days and 7-days senktide injection models. But significant aggravation of dopaminergic degeneration and motor deficit were only observed in 7-days senktide-treated group. This observation may imply that the effect of senktide in 6-OHDA-lesioned model was a time-dependent process. Induction of NR1, JNK, and c-Jun phosphorylation may be the upstream events induced by early senktide treatment prior to dopaminergic degeneration. Therefore, in our model, no significant deterioration was revealed in 2-days senktide-treated group, although up-regulation of phosphorylated NR1 and JNK pathway molecules were still observed.
In conclusion, our results demonstrated novel in vivo pieces of evidences on the roles of intervention of NK3 peptide in 6-OHDA degeneration model. NK3 peptide could exacerbate neuronal degeneration through modulation of NR1 activities and downstream JNK pathway. A hypothetical cellular mechanism model of NK3 receptor and its agonist is illustrated in Fig. 7. The present data strongly suggested that NK3 peptide would play a critical role in dopaminergic degeneration in PD. For further studies, other intracellular mechanisms related to neuronal degeneration could be examined. In addition, regulation of other NMDA receptor subunit could be investigated to further confirm the effect of senktide.
This work was supported by Mini-AoE project RC/AOE/08-09/02, Research Committee, Hong Kong Baptist University; and HKBU 212607 and HKBU 262210, Research Grants Council, Hong Kong SAR Government. The authors have no conflicts of interest to declare.