Bone cancer pain, one of the most common types of chronic pain, has a strong effect on cancer patients’ quality of life. However, the mechanism and treatment of bone cancer pain has remained elusive.1,2 Current therapies for cancer pain management are not fully effective or have significant dose-limiting adverse effects.3 Therefore, a novel and effective therapeutic approach is urgently needed to deal with this situation.
The N-methyl-D-aspartate receptor (NMDAR), specifically NR2B subunit-dependent synaptic plasticity, plays an essential role in central sensitization4,5 and underlies the central mechanisms of bone cancer pain.6,7 Intrathecal administration of NMDAR antagonists, such as ketamine,8 and NR2B-selective antagonists, such as ifenprodil and Ro25-6981,7,9 demonstrated a potent analgesic effect in animal models of bone cancer pain. However, these antagonists have limited utility in clinical patients because activation of NMDAR is essential for many key physiological functions, and pharmacological inhibition of NMDAR may cause numerous intolerable side effects, such as memory impairment and psychotomimetic effects.10,11 Thus, exploring novel ways to modulate NMDAR without affecting basal receptor activity may be a better analgesic strategy.
Activation of NMDAR induces Ca2+ influx. Increased intracellular Ca2+ binds to calmodulin to activate downstream effectors and to trigger a signaling cascade, which includes the activation of neuronal nitric oxide synthase (nNOS), resulting in an increased nitric oxide (NO) production in the spinal cord.12 NO is an important neurotransmitter contributing to the central and peripheral sensitization in both inflammatory and neuropathic pain.13–16 nNOS, producing NO in neurons, also contributes to spinal nociceptive processing. Peripheral inflammation and nerve injury upregulate spinal nNOS expression.17–21 Administration with selective nNOS inhibitors, or targeted disruption of the nNOS gene, remarkably attenuated pain hypersensitivity in inflammatory and neuropathic pain.22–24
NMDAR-mediated nNOS activation is facilitated by scaffolding protein postsynaptic density protein-95 (PSD-95), which attaches NMDAR to intracellular signaling molecules nNOS at neuronal synapses via its second N-terminal PSD-95/Dlg/ZO-1 (PDZ) domain.12,25–28 This binding couples NMDAR activity to the production of NO, which mediates NMDAR-dependent central sensitization.
It has been reported that activation of NMDAR is unaffected by genetically disrupting PSD-95 in vivo29 or by suppressing PSD-95 expression in vitro.12 Therefore, perturbing the NMDAR/PSD-95/nNOS interaction, which dissociates NMDAR activity from NO production and suppresses central sensitization, should be an alternative therapeutic approach in pain treatment without interfering with physiological function of NMDAR ion channel. Current evidence suggests that perturbing nNOS/PSD-95 or PSD-95/NMDAR interactions inhibited spinal dorsal horn hyperexcitability and reversed hyperalgesia induced by inflammation and nerve injury.30–32
However, whether inhibition of NMDAR/PSD-95 interaction could relieve bone cancer pain has not been reported. We investigated the hypothesis that perturbing the specific interaction between spinal NR2B-containing NMDAR and PSD-95 could suppress central sensitization and subsequently attenuate bone cancer-related pain behaviors. This might be achieved by intrathecal injection of peptides that bind to PDZ2 domains. Therefore, a peptide comprising the 9 COOH-terminal residues of NR2B (Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; NR2B9c)27 was constructed to competitively bind PDZ2 domains. The changes of bone cancer-related pain behaviors in mice and the expression of NR2B phosphorylation at Tyr1472 and nNOS in the spinal cord were then evaluated.
Experiments were approved by the Institutional Animal Care and Use Committee at the Medical School of Nanjing University and were in accordance with the guidelines for the use of laboratory animals.33 All efforts were made to minimize animal suffering and to reduce the number of animals used in this study. Experiments were performed on male C3H/HeJ mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China; SCXK JING 2000-0009), 4 to 6 weeks old, weighing 20 to 25 g. The mice were housed in groups of 5/cage, fed with food pellets, and provided with water ad libitum. All animals were maintained in a temperature-controlled (21°C ± 1°C) room with 12-hour alternating dark/light cycles.
Cell Culture and Implantation
Osteosarcoma NCTC 2472 cells (American Type Culture Collection, ATCC, 2087787) were incubated and subcultured in NCTC 135 medium (Sigma-Aldrich, St. Louis, MO) with 10% horse serum (Gibco, Grand Island, NY) at 37°C in an atmosphere of 5% CO2 and 95% air (Thermo Forma, Marietta, OH) and passaged twice a week according to the recommendations provided by ATCC.
The mouse model of bone cancer pain was generated as previously described by Schwei et al.34 On day 0, mice were anesthetized with an intraperitoneal injection of 50-mg/kg pentobarbital sodium (1% in physiologic saline), and a superficial incision was made in the skin above the right articulatio genu using eye scissors. Gonarthrotomy was performed, which exposed the femur condyles. A light depression was made using a dental bur. A 30-gauge needle was used to perforate the cortex, and a 25-μL microsyringe was used to inject a volume of 20-μL α-minimum essential medium (α-MEM) containing no or 2 × 105 NCTC 2472 cells into the intramedullary space of the femur, which corresponded to sham or tumor-bearing mice, respectively. Subsequently, the injection hole was sealed using dental amalgam, followed by copious irrigation with normal saline. The wound was then sutured closed.
Drug Preparation and Intrathecal Injection
Myr-NR2B9c peptide comprises the final 9 amino acids of the NR2B subunit cytoplasmic tail (KLSSIESDV), including the PSD-95 PDZ domain-binding motif (ESDV). This sequence is conjugated to a myristic acid at its N-terminal end for intracellular delivery. A “negative control” peptide, Myr-NR2BAA, was synthesized with a double substitution in the PDZ domain-binding motif (EADA), thereby rendering it not able to bind its target and being inactive. Peptides were synthesized using a solid-phase peptide synthesizer, and the purity of conjugates (>95%) were assayed by high-performance liquid chromatography and mass spectrometry (GL Biochem, Shanghai, Ltd., China).
Myr-NR2B9c (GL Biochem, China, P110921-MJ264459) or its isomer Myr-NR2BAA (GL Biochem, China, P110921-MJ264546) was dissolved in dimethylsulfoxide (DMSO) and then diluted in physiologic saline to a final concentration of 0.2 mg/mL (the concentration of DMSO was 10%, v/v). For vehicle treatment, 10% DMSO was used. At day 14 after the operation, in the Myr-NR2B9c treatment group, Myr-NR2B9c (1 μg/5 μL) was injected intrathecally, and in the Myr-NR2BAA treatment group, Myr-NR2BAA (1 μg/5 μL) was injected intrathecally. The control group received the same volume of vehicle injection.
Intrathecal injections were performed manually between the L5 and L6 lumbar space in unanesthetized mice according to a previous method described by Hylden and Wilcox.35 The injection was performed using a 25-gauge needle attached to a glass microsyringe. Each animal was injected with a volume of 5 μL. The accurate placement of the needle was confirmed by a quick “flick” of the mouse’s tail.
The study included 5 independent experiments, and different mice were used in each experiment.
Experiment 1: Pain Behaviors Over Time
The time course of changes in pain behaviors after NCTC 2472 cells inoculation was examined. Withdrawal threshold and latency to mechanical and thermal stimulation, respectively, were examined during a 2-week period: day −1 before the operation and days 3, 5, 7, 10, and 14 after the operation in both tumor-bearing mice (n = 8) and sham mice (n = 8) groups.
Experiment 2: Measurement of NR2B Phosphorylation at Tyr1472, PSD-95 and nNOS Expression in the Spinal Cords of Tumor-Bearing Mice
To determine whether bone cancer altered the expression of phospho-Tyr1472 NR2B, PSD-95, and nNOS in the L3-L5 spinal cord, tissue samples were obtained from tumor-bearing mice on days −1, 5, 7, 10, 14 and from sham mice on day 14 after the inoculation for Western blotting analyses (n = 3/group).
Experiment 3: Effects of Intrathecal Injection of Myr-NR2B9c on Protein Interactions Between NR2B Subunit and PSD-95 in the Spinal Cord
To determine whether Myr-NR2B9c disturbed the protein interactions between NR2B and PSD-95 in the spinal cord, 3 groups of mice (n = 3) were used, which included Myr-NR2B9c treatment group, Myr-NR2BAA treatment group, and control group and then co-immunoprecipitation was applied.
Experiment 4: Effects of Intrathecal Injection of Myr-NR2B9c on Bone Cancer-Related Pain Behaviors
Twenty-four mice were randomly divided into 3 groups (n = 8): tumor-bearing mice receiving Myr-NR2B9c (1 μg/5 μL), tumor-bearing mice receiving Myr-NR2BAA (1 μg/5 μL), and tumor-bearing mice receiving vehicle (10% DMSO). After pain behaviors were observed on day 14 after inoculation, the mice were intrathecally administered either peptide or vehicle. Pain behaviors were measured at 2, 4, 6, 8, 10, 12, and 24 hours after administration. The data measured before administration were regarded as baseline data.
Experiment 5: Effects of Intrathecal Injection of Myr-NR2B9c on the Expression of Phospho-Tyr1472 NR2B, PSD-95 and nNOS in the Spinal Cord
To determine whether Myr-NR2B9c altered the expression of phospho-Tyr1472 NR2B, PSD-95, and nNOS in the L3-L5 spinal cord, tissue samples were obtained from mice of each group before intrathecal injection and at 2, 4, 6, 8, 10, 12, and 24 hours after intrathecal injection for Western blotting analyses (n = 3/group).
All tests were performed during the light phase. Before each test, mice were allowed to acclimatize for at least 30 minutes. All behavioral responses were measured by the experimenters who were blind to the treatment groups.
Mechanical allodynia was assessed using von Frey filaments (Stoelting, Wood Dale, IL), which were applied to the right hindpaw according to our previous study.7 This method has been previously described by Chaplan et al.36 and was modified to assess pain in mice. Mice were placed into individual transparent plexiglass compartments (10 × 10 × 15 cm) onto a metal mesh floor (graticule: 0.5 × 0.5 cm). Paw withdrawal mechanical threshold (PWMT) was measured using a set of von Frey filaments (0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g). Filaments were pressed vertically against the plantar surface with sufficient force to cause a slight bending against the paw and were held for 6 to 8 seconds with a 10-minute interval between stimulations. Brisk withdrawal or paw flinching were considered positive responses. Each mouse was tested 5 times per stimulus strength. The lowest von Frey filament, which had 3 or more positive responses, was regarded as the PWMT.
Thermal hyperalgesia to radiant heat was determined according to a previous method described by Hargreaves et al.37 Mice were placed into individual transparent plexiglass compartments (10 × 10 × 5 cm) onto a 3-mm-thick-glass floor. A radiant thermal stimulator (BME410A, Institute of Biological Medicine, Academy of Medical Science, China) was focused onto the plantar surface of the hindpaw through the glass floor. The nociceptive end points in the radiant heat test were the characteristic lifting or licking of the hindpaw, and the time to the end point was considered the paw withdrawal thermal latency (PWTL). A cutoff time of 20 seconds was used to avoid tissue damage. There were 5 trials per mouse and 5-minute intervals between 2 trials. The mean PWTL was obtained from the latter 3 stimuli.
Western Blotting Analyses
Mice were deeply anesthetized with sevoflurane and killed by decapitation. Spinal cord L3-L5 segments were removed rapidly and stored in liquid nitrogen. Tissue was subsequently homogenized in lysis buffer. After being placed on ice for 30 minutes, the homogenate was centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was collected and assayed for protein concentrations using BCA Protein Assay Kit (KeyGEN Biotech, Nanjing, China) and stored at −70°C. Loading buffer was added to protein lysates (50 μg), and samples were boiled at 100°C for 5 minutes. Protein samples were separated using SDS-PAGE (8%) (Bio-Rad, Hercules, CA) and subsequently transferred to polyvinylidene difluoride membranes (Millopore Corporation, MA). Filter membranes were blocked with 5% nonfat milk for 2 hours at room temperature and then incubated with the following primary antibodies, rabbit anti-phospho-Tyr1472 NR2B (1:500, CST, Biotechnology), rabbit anti-PSD-95 (1:500; Biotechnology, Santa Cruz, CA), and rabbit anti-nNOS (1:1000; Abcam, United Kingdom) at 4°C overnight. The membrane was then washed with TBST buffer and incubated with secondary antibody conjugated with horseradish peroxidase (1:5000; BioVision, CA) for 2 hours at room temperature. The immune complexes were detected using the ECL system (Santa Cruz Biotechnology, CA) and visualized on Kodak BioMax MR X-ray film (Kodak, New York, NY). β-actin was used as a loading control. Images of the Western blot protein bands were collected and analyzed using Quantity One V4.40 (Bio-Rad). Data are presented as the mean ± SD.
Three additional groups of naive mice were intrathecally injected with Myr-NR2B9c (1 μg), Myr-NR2BAA (1 μg), or vehicle, 30 minutes before being killed and before dissection of tissue under sevoflurane anesthesia. Five microgram rabbit anti-NR2B antibody (Sigma) was incubated with 100 μL protein A-Sepharose slurry for 1 hour, and the complex was spun down at 2000 rpm for 4 minutes. The lumbar spinal cord lysate (500 μg) of mice from different groups as mentioned above, collected similar to Western blotting, was then added to the Sepharose beads, and the mixture was incubated for 2 to 3 hours at 4°C. The mixture was washed 4 times in immunoprecipitation buffer (1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.57 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin in phosphate-buffered saline, and pH 7.4). The proteins were separated using SDS-PAGE and detected using NR2B or PSD-95 antibody. As a positive control (Lys), 50 μg normal lumbar spinal cord lysate samples was loaded onto the gel.
Data are expressed as the mean ± SD (standard deviation). Animals were assigned to different treatment groups in a randomized manner. Changes in PWMT and PWTL after inoculation and administration were compared with basal values, respectively, using a 2-way analysis of variance for repeated measures, followed by Bonferroni correction for between-group comparisons. Western blotting data and co-immunoprecipitation data were analyzed using a 1-way analysis of variance for overall differences among groups followed by Bonferroni correction for between-group comparisons. In multiple comparisons, the Bonferroni correction was used by lowering the critical significance probability to P < 0.05/n, where n equals the number of multiple comparisons in a given analysis. Statistical analysis was performed using SPSS 16.0 software (IBM Corporation, Armonk, NY). All reported P values for paired comparisons are post hoc corrected P values. The P value < 0.01 was considered statistically significant.
Pain Behavior Overtime
There was no significant difference in PWMT or PWTL between the tumor-bearing mice and sham mice before operation. Compared with the basal value before inoculation, in response to the operation of both tumor-bearing mice (P < 0.0001) and sham mice (P = 0.0003), the ipsilateral hindpaw showed a significant decrease in PWMT in response to von Frey filament stimulation on day 3. There was no significant difference in PWMT on day 5 (P = 0.99 in both group tumor and sham) when compared with the basal value before inoculation. Tumor-bearing mice then showed a decrease in PWMT of the ipsilateral hindpaw on day 7 (P = 0.001) that gradually decreased over time until day 14 (P = 0.0004). In addition, compared with group Sham, PWMT in group tumor was significantly decreased on day 7 (P = 0.0002), day 10 (P < 0.0001), and day 14 (P < 0.0001) (Fig. 1A).
Compared with the basal value before inoculation, the right hindlimb of both tumor-bearing (P = 0.003) and sham mice (P = 0.005) showed a significant decrease in PWTL in response to radiant heat stimulation on day 3. There was no significant difference in PWTL on day 5 (P = 0.99 in both group tumor and sham) when compared with the basal value before inoculation. PWTL of the tumor-bearing mice on day 10 (P = 0.001) showed a significant decrease and gradually decreased along with the development of bone cancer pain until day 14 (P = 0.0001). Compared with group Sham, PWTL in group tumor was significantly decreased on day 10 (P < 0.0001) and day 14 (P < 0.0001) (Fig. 1B). In addition, no significant difference in PWTL or PWMT was observed in sham mice between days 5, 7, 10, 14 and day −1. And the transient decrease of PWMT and PWTL on day 3 could be attributed to the arthrotomy.
Measurement of Expression of Phospho-Tyr1472 NR2B, PSD-95, and nNOS in the Spinal Cord of Tumor-Bearing Mice
The expression levels of spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95 were significantly upregulated in tumor-bearing mice compared with that of sham mice, as revealed by Western blotting analyses (Fig. 2). Compared with that of sham mice on day 14 and preoperative levels, spinal phospho-Tyr1472 NR2B expression levels increased on day 5 (P = 0.002 and P = 0.001, respectively) after operation, and this increase persisted on day 7 (P < 0.0001), day 10 (P < 0.0001), and day 14 (P < 0.0001). Compared with that of sham mice on day 14 and preoperative levels, spinal nNOS expression levels increased on day 5 (P < 0.0001) after operation, and this increase persisted on day 7 (P < 0.0001), day 10 (P< 0.0001), and day 14 (P< 0.0001). Compared with sham mice on day 14 and preoperative levels, spinal PSD-95 expression levels increased on day 5 (P < 0.0001) after operation, and this increase persisted on day 7 (P = 0.0002 and P = 0.0006, respectively), day 10 (P < 0.0001), and day 14 (P < 0.0001). No significant differences in the expression levels of the 3 proteins were observed between sham mice and preoperative levels.
Myr-NR2B9c Disrupts Protein Interactions Between NR2B-Containing NMDAR and PSD-95 in Spinal Cord
Co-immunoprecipitation assay was used to detect whether NR2B-PSD-95 interactions were disrupted by intrathecal injection of Myr-NR2B9c. In a separate experiment, after mice were pretreated via intrathecal injection with Myr-NR2B9c (1 μg), Myr-NR2BAA (1 μg), or vehicle (10% DMSO), respectively, NR2B antibody was used to immunoprecipitate NR2B and its interacting proteins from spinal cord homogenates. Co-immunoprecipitation of PSD-95 with NR2B in mice pretreated with Myr-NR2B9c was significantly decreased compared with that in mice pretreated with vehicle (P = 0.00016) (Fig. 3). There was no significant difference in PSD-95/NR2B between mice pretreated with Myr-NR2BAA and mice pretreated with vehicle (P = 0.962).
Effects of Intrathecal Administration of Myr-NR2B9c on Pain Behaviors Induced by Bone Cancer
Compared with the base levels before administration and compared with those levels of tumor-bearing mice receiving vehicle at the same time point, both PWMT and PWTL of tumor-bearing mice receiving Myr-NR2B9c were ameliorated after intrathecal administration. Two hours after administration, PWMT of mice receiving Myr-NR2B9c was significantly increased compared with mice receiving vehicle (P = 0.001). Four hours later, PWMT was (1.28 ± 0.41) g (P < 0.0001), and the difference was maintained until 10 hours after administration (P = 0.004). Meanwhile, 2 hours after administration, PWTL was significantly increased to (14.33 ± 2.05) seconds (P = 0.007), and the increase continued to 10 hours after administration (P = 0.009). The antihyperalgesic efficacy of Myr-NR2B9c began 2 hours after administration, achieved maximal levels at 4 hours, attenuated at 10 hours, and disappeared at 24 hours. No significant difference in PWMT or PWTL was observed in tumor-bearing mice between the baseline level and the level examined at any time point after administration of Myr-NR2BAA or vehicle (Fig. 4).
Effects of Intrathecal Administration of Myr-NR2B9c on the Expression Levels of Phospho-Tyr1472 NR2B, nNOS, and PSD-95 in Spinal Cord
Intrathecal injection of Myr-NR2B9c significantly decreased the level of tyrosine phosphorylation of NR2B at Tyr1472 in spinal cord 2 hours after administration compared with preadministration baseline on day 14 after operation (P < 0.0001). The decrease continued to exist at 10 hours after drug administration (P = 0.003). Compared with preadministration baseline, the expression level of nNOS in spinal cord was significantly downregulated at 6 hours (P < 0.0001) and 8 hours (P = 0.004) after intrathecal injection. Meanwhile, the expression of spinal PSD-95 was also downregulated at 2 hours after administration of Myr-NR2B9c (P = 0.0002). However, there was no significant difference in the expression level of PSD-95 between 4, 6, 8, 10, 12, 24 hours after administration and baseline before administration (Fig. 5).
The present study showed that hyperalgesia and allodynia induced by bone cancer in a mouse model is associated with upregulation of phospho-Tyr1472 NR2B, PSD-95, and nNOS in the spinal cord. Binding of PSD-95 to NMDARs in the spinal cord, containing specifically NR2B subunits, can facilitate downstream signaling of NMDAR activation, contributing to NMDAR-dependent central sensitization. Selectively perturbing such interactions with the mimetic peptide, Myr-NR2B9c, reduces abnormal pain-related behaviors and also downregulates expression levels of phospho-Tyr1472 NR2B, PSD-95, and nNOS in the spinal cord. In particular, our findings provided insight into the important role of the specific physical interaction between spinal NR2B-containing NMDAR and PSD-95 in the maintenance of bone cancer pain.
Bone cancer pain, which exhibits inflammatory, neuropathic, and tumorigenic characteristics,38 remains a clinically challenging problem to treat effectively. It is now accepted that NMDAR especially NR2B subunit-dependent synaptic plasticity in pain pathways contributes to central sensitization, facilitating bone cancer pain.4,7,9 PSD, an important structure at excitatory synapses of central neurons, contains tight signaling complexes of multiproteins (such as inotropic glutamate receptors, scaffolding proteins, and so on), which are well positioned to coordinate postsynaptic signal transduction with activity-dependent changes in postsynaptic structure and function. The major constituent of PSD, PSD-95, mediates protein interactions and plays a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. Numerous studies suggest an important role for PSD-95 in synaptic plasticity in various pain models.39–43 The second PDZ domain of PSD-95 can selectively bind NR2B subunits of NMDAR, which contain a PDZ-binding motif (tSXV) at the C-terminus.27 This interaction couples the NMDAR-mediated Ca2+ influx to intracellular effector proteins and signaling enzymes, such as nNOS, modulates membrane receptor stability, and inhibits internalization of NMDAR,44 contributing to NMDAR-dependent central sensitization.
In the present study, we demonstrated that perturbing PSD-95 interactions with NR2B-subtype receptors, using a peptide-mimetic strategy, could attenuate bone cancer pain in mice. First, similar to our previous investigations,7,45,46 the progressive mechanical allodynia and thermal hyperalgesia induced by implantation of sarcoma cells into the right femur in the present study (Fig. 1) indicated successful establishment of a mouse model of bone cancer pain. Meanwhile, this aggravation of pain behaviors was accompanied by a notable upregulation of phospho-Tyr1472 NR2B, PSD-95, and nNOS (Fig. 2), which implicated all 3 proteins might be involved in the development and maintenance of bone cancer pain. Next, results of co-immunoprecipitation (Fig. 3) revealed that intrathecal administration of Myr-NR2B9c, in which the COOH-terminal tSXV motif mimics residues unique to the NR2B COOH-terminus, selectively reduced the co-immunoprecipitation of PSD-95 with NR2B in the spinal cord. Intrathecal administration of Myr-NR2BAA, in which the COOH-terminal tSXV motif contains a double point mutation rendering it incapable of binding PSD-95, did not affect the physical interaction between PSD-95 and NR2B. Our study further showed that a single intrathecal administration of Myr-NR2B9c on day 14 after inoculation significantly attenuated bone cancer-induced mechanical allodynia and thermal hyperalgesia, while the control peptide, Myr-NR2BAA, had no effect at all. The antihyperalgesic efficacy of Myr-NR2B9c began at 2 hours after drug administration, was maximal at 4 hours, attenuated at 10 hours, and disappeared at 24 hours (Fig. 4). Combining the results of coimmunoprecipitation, we speculate that Myr-NR2B9c might exert its analgesic effect on the bone cancer pain model via disrupting the NR2B-PSD-95 interaction in the spinal cord. Moreover, results of Western blotting showed that intrathecal administration of Myr-NR2B9c also downregulated spinal phospho-Tyr1472 NR2B, PSD-95, and nNOS expression, which corresponded with its behavioral effects (Fig. 5).
The results of the present study are consistent with previous findings. D’Mello et al.’s31 study showed that intrathecal injection of Tat-NR2B9c in rats prevents neuronal plasticity and attenuates behavioral signs of mechanical and cold hypersensitivity in neuropathic pain. The contribution of PSD-95-mediated protein interactions to pain was also indicated through the use of alternative peptides, Tat-PSD-95 PDZ232 and Tat-nNOS (residues 1–299),30 that mimic the second PDZ domain of PSD-95 and PSD-95 binding domain of nNOS, respectively. Both peptides exhibit analgesic effects through disruption of NMDAR-PSD-95 interaction or nNOS-PSD-95 interaction in inflammatory pain or neuropathic pain models. In addition, a cyclic peptide, CN2097, that blocks interactions at all PDZ domains of PSD-95 has also been shown to block central sensitization and attenuate thermal hyperalgesia after nerve injury.47
Several studies using NR2B9c may indicate potential downstream signaling mechanisms by which perturbing PSD-95 interaction with NR2B reduces nociceptive hypersensitivity. Therefore, we speculate that intrathecal injection of Myr-NR2B9c may exert analgesic effects mainly via promoting NR2B internalization and inhibiting nNOS activation.
The first analgesic mechanism is that NR2B9c may interfere with membrane insertion and promote rapid internalization of NMDAR. The internalization of NMDAR is mediated by Tyrosine-based internalization motifs, YEKL, on the distal C-terminus of NR2B. YEKL motifs bind to AP-2 adaptor complexes, thereby linking cargo to clathrin and regulating their inclusion to clathrin-coated vesicles, and ultimately sorting receptors toward degradation or recycling pathways.48 Once NR2B is phosphorylated at the tyrosine 1472 site by Src-family tyrosine kinases, such as Src44 and Fyn,49 it cannot bind to μ subunit of AP-2, thereby the internalization of NMDAR is inhibited.50 Accumulated evidence has indicated that Tyr1472 phosphorylation, which is important for stable surface expression of NMDAR at synaptic sites, plays a key role in synaptic plasticity, induction of long-term potentiation and central sensitization.49 It has been reported that tyrosine phosphorylation of NR2B at Tyr1472 in the spinal dorsal horn contributes to development of opioid-induced hyperalgesia, as well as inflammatory and neuropathic pain.6,51–53 PSD-95, which assembles Src and Fyn around NR2B via its multiple domains, allowing the tyrosine phosphorylation of NR2B at Tyr1472, thereby inhibiting the internalization of NMDAR, is involved in the regulation and stability of NMDAR at synaptic sites.54 After administration of Myr-NR2B9c, NR2B-PSD-95 interaction may be disrupted, reducing Tyr1472 phosphorylation and resulting in NR2B-regulated internalization via the YEKL motif on the NR2B C-terminus, thus downregulating overexpression of NMDAR induced by nociceptive stimulus at postsynaptic membrane, inhibiting central sensitization and attenuating pain behaviors. The results of Western blotting in the present study, which indicated a downregulation of phospho-Tyr1472 NR2B from 2 to 10 hours after administration of Myr-NR2B9c, also verifies this hypothesis. Collectively, these studies in combination with our current data would support a role for NMDAR internalization in the analgesic mechanisms of Myr-NR2B9c.
The other important analgesic mechanism is that NR2B9c uncouples NMDAR activation from the downstream nNOS signaling pathway, inhibits nNOS activity, and reduces NO production. nNOS, which is mainly located in the superficial dorsal horn, plays a key role in central sensitization in various pain models.17–24 Nociceptive stimulus causes an augmentation of excitatory synaptic transmission and triggers activation of NMDAR in the spinal cord. Increased intracellular Ca2+ via NMDAR can activate downstream signaling molecules located near the channel pore, such as nNOS, that is physically linked to NMDAR via PSD-95, leading to the over production of NO. Myr-NR2B9c can competitively bind to the second PDZ domain of PSD-95, thus preventing the formation of NR2B/PSD-95/nNOS complex, dissociating NMDAR activation and Ca2+ influx from nNOS activation, reducing NMDAR-mediated spinal plasticity and central sensitization. In addition, previous studies have demonstrated that NR2B9c exerts a neuroprotective effect in ischemic brain damage via preventing NMDAR-dependent generation of NO, thereby alleviating nervous excitotoxicity.55,56
In addition, several details in our study need to be explained. First, Myristic acid has high hydrophobicity that enables its incorporation into the phospholipid bilayer membrane57 and has been shown to be effective at delivering attached compounds across cellular membranes.58,59 Therefore, we used myristic acid as an alternative modification for intracellular delivery of the target peptide. Second, NR2B9c has a clear mechanism based on a specific and restricted set of molecular targets. It has been demonstrated, using a proteomic and biochemical analysis of the interactions of NR2B9c with all known human proteins that it may bind, that this compound has high specificity, impacting excitotoxicity solely through its interactions with PSD-95 and with nNOS, and other interactions would remain intact.60 Therefore, NR2B9c may also affect the interaction of PSD-95-nNOS. However, in rat models of inflammatory pain and neuropathic pain, D’Mello et al.31 considered that the antinociceptive effects of NR2B9c are due to a more specific disruption of NR2B and PSD-95 interactions. Some previous work also confirms that when the NMDAR-PSD95 interaction is unaffected, neuroprotection is not achieved.12,55,60 Therefore, we supposed that perturbing NR2B-PSD-95 interaction may be the main mechanism of the antinociceptive effects of NR2B9c. Third, NMDARs are also implicated in many important physiopathologic processes throughout central nervous system. Experiments in vivo and in vitro have demonstrated that perturbing PSD-95 interactions with NR2B exerts analgesic or neuroprotective effects without affecting NMDAR activity or Ca2+ influx.48 Therefore, the adverse consequences of blocking NMDAR could be avoided. Moreover, D’Mello et al.31 reported that NR2B9c alleviated neuropathic pain and did not influence the performance of rats on the rotarod, indicating a lack of adverse locomotor effects that are commonly caused by conventional NMDAR antagonists. Unlike NMDAR antagonists, perturbing interaction between NR2B and PSD-95 maintains glutamatergic signaling. This may be an advantage of NR2B9c over NMDAR blockers, which cause sedation, memory impairment, and psychotomimetic effects in humans. Thus, our present findings may serve as novel and successful analgesic therapy strategies to treat chronic pain and potentially other NMDAR-dependent central nervous system pathologies, reducing side effects associated with NMDAR blockade.
In summary, the present study demonstrated that bone cancer-induced mechanical allodynia and thermal hyperalgesia as well as the upregulation of spinal phospho-Tyr1472 NR2B, PSD-95, and nNOS. Myr-NR2B9c, a mimetic peptide that disrupts interactions specifically between NR2B-containing NMDAR and PSD-95, alleviated the cancer-induced mechanical allodynia and thermal hyperalgesia. More importantly, these results validate the concept that targeting of the NMDAR-PSD-95 interaction could be a practical and novel strategy to selectively prevent NMDAR-dependent phenomena, such as wind-up and central sensitization, without receptor blockade, a likely cause of neurological side effects associated with NMDAR antagonists. Therefore, mimetic peptides, specifically NR2B9c, should be investigated further as novel analgesics for clinical treatment of chronic pain.
Name: Yue Liu, MD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Yue Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xinlong Cui, MD.
Contribution: This author helped conduct the study and collect the data.
Attestation: Xinlong Cui has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Yu-E Sun, MD.
Contribution: This author helped analyze the data.
Attestation: Yu-E Sun has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xuli Yang, MD.
Contribution: This author helped analyze the data.
Attestation: Xuli Yang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Kun Ni, MD.
Contribution: This author helped conduct the study.
Attestation: Kun Ni has seen the original study data, and approved the final manuscript.
Name: Yu Zhou, MD.
Contribution: This author helped collect the data.
Attestation: Yu Zhou has seen the original study data, and approved the final manuscript.
Name: Zhengliang Ma, MD, PhD.
Contribution: This author helped design the study and conduct the study.
Attestation: Zhengliang Ma has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Xiaoping Gu, MD, PhD.
Contribution: This author helped design the study and conduct the study.
Attestation: Xiaoping Gu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Jianren Mao, MD, PhD.
1. Coleman RE. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin Cancer Res. 2006;12:6243s–9s
2. Colvin L, Fallon M. Challenges in cancer pain management–bone pain. Eur J Cancer. 2008;44:1083–90
3. Meuser T, Pietruck C, Radbruch L, Stute P, Lehmann KA, Grond S. Symptoms during cancer pain treatment following WHO-guidelines: a longitudinal follow-up study of symptom prevalence, severity and etiology. Pain. 2001;93:247–57
4. Qu XX, Cai J, Li MJ, Chi YN, Liao FF, Liu FY, Wan Y, Han JS, Xing GG. Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain. Exp Neurol. 2009;215:298–307
5. Woolf CJ, Thompson SW. The induction and maintenance of central sensitization is dependent on N
-methyl-D-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states. Pain. 1991;44:293–9
6. Matsumura S, Kunori S, Mabuchi T, Katano T, Nakazawa T, Abe T, Watanabe M, Yamamoto T, Okuda-Ashitaka E, Ito S. Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472. Eur J Neurosci. 2010;32:798–810
7. Gu X, Zhang J, Ma Z, Wang J, Zhou X, Jin Y, Xia X, Gao Q, Mei F. The role of N
-methyl-D-aspartate receptor subunit NR2B in spinal cord in cancer pain. Eur J Pain. 2010;14:496–502
8. Saito O, Aoe T, Kozikowski A, Sarva J, Neale JH, Yamamoto T. Ketamine and N-acetylaspartylglutamate peptidase inhibitor exert analgesia in bone cancer pain. Can J Anesth. 2006;53:891–8
9. Pedersen LM, Gjerstad J. Spinal cord long-term potentiation is attenuated by the NMDA-2B receptor antagonist Ro 25-6981. Acta Physiol (Oxf). 2008;192:421–7
10. Parsons CG. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol. 2001;429:71–8
11. Chizh BA, Headley PM. NMDA antagonists and neuropathic pain–multiple drug targets and multiple uses. Curr Pharm Des. 2005;11:2977–94
12. Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF, Tymianski M. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science. 1999;284:1845–8
13. Miyamoto T, Dubin AE, Petrus MJ, Patapoutian A. TRPV1 and TRPA1 mediate peripheral nitric oxide-induced nociception in mice. PLoS One. 2009;4:e7596
14. Naik AK, Tandan SK, Kumar D, Dudhgaonkar SP. Nitric oxide and its modulators in chronic constriction injury-induced neuropathic pain in rats. Eur J Pharmacol. 2006;530:59–69
15. Ikeda H, Stark J, Fischer H, Wagner M, Drdla R, Jäger T, Sandkühler J. Synaptic amplifier of inflammatory pain in the spinal dorsal horn. Science. 2006;312:1659–62
16. Cury Y, Picolo G, Gutierrez VP, Ferreira SH. Pain and analgesia: The dual effect of nitric oxide in the nociceptive system. Nitric Oxide. 2011;25:243–54
17. Ma ZL, Zhang W, Gu XP, Yang WS, Zeng YM. Effects of intrathecal injection of prednisolone acetate on expression of NR2B subunit and nNOS in spinal cord of rats after chronic compression of dorsal root ganglia. Ann Clin Lab Sci. 2007;37:349–55
18. Choi JI, Kim WM, Lee HG, Kim YO, Yoon MH. Role of neuronal nitric oxide synthase in the antiallodynic effects of intrathecal EGCG in a neuropathic pain rat model. Neurosci Lett. 2012;510:53–7
19. Lam HH, Hanley DF, Trapp BD, Saito S, Raja S, Dawson TM, Yamaguchi H. Induction of spinal cord neuronal nitric oxide synthase (NOS) after formalin injection in the rat hind paw. Neurosci Lett. 1996;210:201–4
20. Lee JS, Zhang Y, Ro JY. Involvement of neuronal, inducible and endothelial nitric oxide synthases in capsaicin-induced muscle hypersensitivity. Eur J Pain. 2009;13:924–8
21. Chacur M, Matos RJ, Alves AS, Rodrigues AC, Gutierrez V, Cury Y, Britto LR. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection. Braz J Med Biol Res. 2010;43:367–76
22. Handy RL, Moore PK. Effects of selective inhibitors of neuronal nitric oxide synthase on carrageenan-induced mechanical and thermal hyperalgesia. Neuropharmacology. 1998;37:37–43
23. Tanabe M, Nagatani Y, Saitoh K, Takasu K, Ono H. Pharmacological assessments of nitric oxide synthase isoforms and downstream diversity of NO signaling in the maintenance of thermal and mechanical hypersensitivity after peripheral nerve injury in mice. Neuropharmacology. 2009;56:702–8
24. Chu YC, Guan Y, Skinner J, Raja SN, Johns RA, Tao YX. Effect of genetic knockout or pharmacologic inhibition of neuronal nitric oxide synthase on complete Freund’s adjuvant-induced persistent pain. Pain. 2005;119:113–23
25. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell. 1996;84:757–67
26. Sheng M. Molecular organization of the postsynaptic specialization. Proc Natl Acad Sci U S A. 2001;98:7058–61
27. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science. 1995;269:1737–40
28. Christopherson KS, Hillier BJ, Lim WA, Bredt DS. PSD-95 assembles a ternary complex with the N
-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain. J Biol Chem. 1999;274:27467–73
29. Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O’Dell TJ, Grant SG. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature. 1998;396:433–9
30. Florio SK, Loh C, Huang SM, Iwamaye AE, Kitto KF, Fowler KW, Treiberg JA, Hayflick JS, Walker JM, Fairbanks CA, Lai Y. Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents. Br J Pharmacol. 2009;158:494–506
31. D’Mello R, Marchand F, Pezet S, McMahon SB, Dickenson AH. Perturbing PSD-95 interactions with NR2B-subtype receptors attenuates spinal nociceptive plasticity and neuropathic pain. Mol Ther. 2011;19:1780–92
32. Tao F, Su Q, Johns RA. Cell-permeable peptide Tat-PSD-95 PDZ2 inhibits chronic inflammatory pain behaviors in mice. Mol Ther. 2008;16:1776–82
33. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109–10
34. Schwei MJ, Honore P, Rogers SD, Salak-Johnson JL, Finke MP, Ramnaraine ML, Clohisy DR, Mantyh PW. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci. 1999;19:10886–97
35. Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol. 1980;67:313–6
36. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55–63
37. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32:77–88
38. Pan HL, Zhang YQ, Zhao ZQ. Involvement of lysophosphatidic acid in bone cancer pain by potentiation of TRPV1 via PKCε pathway in dorsal root ganglion neurons. Mol Pain. 2010;6:85
39. De Roo M, Klauser P, Mendez P, Poglia L, Muller D. Activity-dependent PSD formation and stabilization of newly formed spines in hippocampal slice cultures. Cereb Cortex. 2008;18:151–61
40. Ehrlich I, Klein M, Rumpel S, Malinow R. PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A. 2007;104:4176–81
41. Tao F, Tao YX, Gonzalez JA, Fang M, Mao P, Johns RA. Knockdown of PSD-95/SAP90 delays the development of neuropathic pain in rats. Neuroreport. 2001;12:3251–5
42. Tao F, Tao YX, Mao P, Johns RA. Role of postsynaptic density protein-95 in the maintenance of peripheral nerve injury-induced neuropathic pain in rats. Neuroscience. 2003;117:731–9
43. Tao YX, Huang YZ, Mei L, Johns RA. Expression of PSD-95/SAP90 is critical for N
-methyl-D-aspartate receptor-mediated thermal hyperalgesia in the spinal cord. Neuroscience. 2000;98:201–6
44. Zhang S, Edelmann L, Liu J, Crandall JE, Morabito MA. Cdk5 regulates the phosphorylation of tyrosine 1472 NR2B and the surface expression of NMDA receptors. J Neurosci. 2008;28:415–24
45. Gu X, Zhou X, Zheng Y, Zhang J, Wang J, Ma Z. Involvement of the spinal NMDA receptor/PKC gamma signaling pathway in the development of bone cancer pain Brain Res. 2010;1335:83–90
46. Ren BX, Gu XP, Zheng YG, Liu CL, Wang D, Sun YE, Ma ZL. Intrathecal injection of metabotropic glutamate receptor subtype 3 and 5 agonist/antagonist attenuates bone cancer pain by inhibition of spinal astrocyte activation in a mouse model. Anesthesiology. 2012;116:122–32
47. LeBlanc BW, Iwata M, Mallon AP, Rupasinghe CN, Goebel DJ, Marshall J, Spaller MR, Saab CY. A cyclic peptide targeted against PSD-95 blocks central sensitization and attenuates thermal hyperalgesia. Neuroscience. 2010;167:490–500
48. Groc L, Choquet D. AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue Res. 2006;326:423–38
49. Nakazawa T, Komai S, Tezuka T, Hisatsune C, Umemori H, Semba K, Mishina M, Manabe T, Yamamoto T. Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N
-methyl-D-aspartate receptor. J Biol Chem. 2001;276:693–9
50. Bonifacino JS, Dell’Angelica EC. Molecular bases for the recognition of tyrosine-based sorting signals. J Cell Biol. 1999;145:923–6
51. Gu X, Wu X, Liu Y, Cui S, Ma Z. Tyrosine phosphorylation of the N
-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine. Mol Pain. 2009;5:76
52. Abe T, Matsumura S, Katano T, Mabuchi T, Takagi K, Xu L, Yamamoto A, Hattori K, Yagi T, Watanabe M, Nakazawa T, Yamamoto T, Mishina M, Nakai Y, Ito S. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. Eur J Neurosci. 2005;22:1445–54
53. Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, Ren K. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci. 2002;22:6208–17
54. Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ. Molecular determinants of NMDA receptor internalization. Nat Neurosci. 2001;4:794–802
55. Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science. 2002;298:846–50
56. Sun HS, Doucette TA, Liu Y, Fang Y, Teves L, Aarts M, Ryan CL, Bernard PB, Lau A, Forder JP, Salter MW, Wang YT, Tasker RA, Tymianski M. Effectiveness of PSD95 inhibitors in permanent and transient focal ischemia in the rat. Stroke. 2008;39:2544–53
57. Farazi TA, Waksman G, Gordon JI. The biology and enzymology of protein N-myristoylation. J Biol Chem. 2001;276:39501–4
58. Ourth DD. Susceptibility in vitro
of Epstein-Barr Virus to myristoylated-peptide. Peptides. 2010;31:1409–11
59. Ourth DD. Antitumor cell activity in vitro
by myristoylated-peptide. Biomed Pharmacother. 2011;65:271–4
60. Cui H, Hayashi A, Sun HS, Belmares MP, Cobey C, Phan T, Schweizer J, Salter MW, Wang YT, Tasker RA, Garman D, Rabinowitz J, Lu PS, Tymianski M. PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors. J Neurosci. 2007;27:9901–15