Treating neuropathic pain is a major clinical challenge, and several key molecules associated with nociception have been suggested as potential targets for novel analgesics. Many studies have reported the anti-nociceptive effects of glial cell-derived neurotrophic factor (GDNF), but the underlying mechanism remains largely unknown. The present study was performed to assess the effects of GDNF in a mouse model of chronic constriction injury (CCI)-induced neuropathic pain. We also determined the potential role of E-cadherin/p120 catenin (p120ctn) signaling in these effects. Mice received an intrathecal acute injection of PBS, GDNF, and DECMA-1 (an E-cadherin functional blocking antibody) or a combination of DECMA-1 with GDNF on the testing days. Our results demonstrated that CCI caused a rapid decrease in E-cadherin and membrane-associated p120ctn in the spinal dorsal horn. Together, these data demonstrated that E-cadherin-associated p120ctn was upregulated by GDNF and that this upregulation was inhibited by pre-treatment with DECMA-1. Moreover, DECMA-1 significantly inhibited the effect of GDNF on thermal hyperalgesia. These data suggest that GDNF might have a therapeutic potential for the treatment of CCI-induced neuropathic pain and that the E-cadherin/p120ctn might play a role in GDNF-induced attenuation of thermal hyperalgesia.
Neuropathic pain is a direct consequence of a lesion or disease affecting the somatosensory system at the peripheral or central level (Haanpaa et al. 2011; Treede et al. 2008) and is characterized by spontaneous pain, hyperalgesia, allodynia, and paresthesia. However, the mechanisms underlying neuropathic pain remain poorly understood; therefore, effective treatment remains a challenge. Existing treatments only provide pain relief for specific causes, which might partially explain the failure to obtain complete pain relief in neuropathic pain conditions. Experimental animal models of neuropathic pain have been used and have yielded a variety of possible therapeutic strategies, including the use of neurotrophic factors (Woolf and Mannion 1999).
Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-β family (TGF-β) (Meng et al. 2000). GDNF was originally identified as a neurotrophic factor for midbrain dopaminergic neurons (Lin et al. 1993) and was considered to be a potential therapeutic for Parkinson’s disease (Glavaski-Joksimovic et al. 2010; Love et al. 2005). GDNF also exerts trophic effects on several types of neurons (Arenas et al. 1995; Oppenheim et al. 1995; Uzdensky et al. 2013). Recently, increasing evidence has shown that GDNF exerts potent analgesic effects on hyperalgesia in rodent neuropathic pain models (Boucher et al. 2000; Dong et al. 2006; Shi et al. 2011; Yagasaki et al. 2013). In addition, immunocytochemistry revealed the localization of GDNF in the superficial layers of spinal cord, an area closely related to pain transmission (Fang et al. 2003; Jongen et al. 1999). There is also evidence of the persistent downregulation of GDNF in the dorsal half of the arthritic rat spinal cord. This downregulation correlated negatively with pain test scores over 8 weeks, suggesting a link between GDNF and pain (Fang et al. 2003). Although GDNF could alleviate neuropathic pain, the mechanisms behind the analgesic effects of GDNF remain unclear. Several studies have assessed the analgesic mechanisms of GDNF. For example, the mechanisms may involve sodium channels, purinergic receptors, and several neuropeptides (Boucher et al. 2000; Charbel Issa et al. 2001; Wang et al. 2003; Xu et al. 2013).
To transmit its intracellular signals, GDNF binds to its high-affinity receptor GFRα1, which activates the receptor tyrosine kinase (RET) (Airaksinen and Saarma 2002). GDNF can also bind to neural cell adhesion molecule (NCAM) via GFRα1 to transmit intracellular signals in a RET-independent fashion (Paratcha et al. 2003). Because NCAM is a cell adhesion molecule, it is possible that E-cadherin, another cell adhesion protein, plays a role in the biological functions of GDNF. Patil et al. demonstrated that E-cadherin is upregulated by GDNF in an L5 spinal nerve transection model (Patil et al. 2011). However, whether E-cadherin signaling mediates the analgesic effects of GDNF is unclear.
E-cadherin is a transmembrane cell surface glycoprotein that mediates calcium-dependent homotypic cell–cell adhesion via its extracellular domains. It is regulated by a variety of biological processes including the maintenance of development and the plasticity of synaptic connectivity (Fiederling et al. 2011; Huntley 2002). Previous studies revealed that E-cadherin is an integral component of the mature synaptic junction complex in the spinal cord (Brock et al. 2004; Seto et al. 1997). However, synapse maturation mainly depends on the cell adhesion functions of cadherin and catenin. Additional studies indicated that the cadherin-associated protein p120 catenin (p120ctn) is required for synapse development and the mature synaptic junction complex. The absence of p120ctn dramatically reduces the density of spines and the synapses formed on their dendrites (Ishiyama et al. 2010). p120ctn is a catenin that binds directly to the juxtamembrane domain (JMD) of classical cadherins, suggesting a role in regulating cell–cell adhesion (Petrova et al. 2012). The activation of cadherin signaling is associated with the recruitment of p120ctn to the JMD to medicate tight adhesion. The present study was performed to determine the effect of GDNF on a mouse model of neuropathic pain and to examine the possible involvement of E-cadherin/p120ctn signaling in this effect.
Materials and Methods
Animals, Drugs, and Drug Administration
Adult male C57BL/6 mice (20–25 g) were obtained from the Experimental Animal Center, Xuzhou Medical College. Mice were housed under a 12-h light/dark cycle with free access to food and water. The care and treatment of all animals was in strict accordance with protocols approved by the Animal Care and use Committee of Xuzhou Medical College (Xuzhou, Jiangsu Province, China) and according to the Declaration of National Institutes of Health Guide for Care and Use of Laboratory Animals (publication no. 80–23, revised 1996). GDNF was purchased from R&D Systems Inc. (Minneapolis, MN, USA). DECMA-1 was obtained from Millipore. Control IgG was purchased from Southern Biotech. GDNF, DECMA-1, and control IgG were all dissolved in PBS (0.2 μg/μL). All drug doses were selected based on previous reports and our preliminary experiments. All drugs were delivered intrathecally (i.t.) in a volume of 5 μL per mouse daily from day 1 after CCI. DECMA-1, an E-cadherin blocking antibody, and control IgG were administered 30 min before GDNF. For i.t. drug delivery, a 2-cm longitudinal skin incision was made above vertebrae L5 and L6. A polyethylene tube (outer diameter 0.6 mm) with the metal wire inside was pushed into one side of the L5–L6 processes at an angle of ~20° to 30° above the vertebral column and 20°–30° to the midline of the vertebra. The catheter was then pushed through the intervertebral space and dura. When signs of dural penetration (sudden movement of the tail or hindlimb) were observed, the guide wire was withdrawn ~2 cm to avoid spinal damage by the metal wire. The catheter was then pushed gently upwards to reach L4 at the lumbar enlargement. The first bead was then fixed by suturing it into the superficial muscle. Another skin incision was then made at the neck area; the catheter was then tunneled under the skin and pulled out of the cut at the neck, where the second bead was fixed into the muscle. The outer end of the catheter was sealed by melting. Heat melting was also used after each injection during the experiment.
Neuropathic Pain Model: Chronic Constrictive Injury
The CCI model was performed as described previously (Bennett and Xie 1988). Briefly, mice were anesthetized using 40-mg/kg sodium pentobarbital (intraperitoneal (i.p.) injection). The left common sciatic nerve was exposed in the left mid-thigh and then loosely ligated using 5-0 silk thread (spaced at a 1-mm interval). Animals in the sham group received surgery identical to that described in CCI, but without nerve injury.
Measurement of Thermal Hyperalgesia
Thermal hyperalgesia was measured using an IITC Plantar Analgesia Meter (IITC Life Science Inc., Woodland Hills, CA, USA) to measure paw withdrawal latency as described previously (Hargreaves et al. 1988). Briefly, each animal was placed in a box containing a smooth, temperature-controlled glass floor. The heat source was focused on a portion of the hindpaw, which was flushed against the glass, and a radiant thermal stimulus was delivered to that site. The stimulus shut off when the hindpaw moved, or after 20 s to prevent tissue damage. The time from the onset of radiant heat to the endpoint was the paw withdrawal latency (PWL). The radiant heat intensity was adjusted to obtain a basal PWL of 10–12 s. Thermal stimuli were delivered three times to each hindpaw at 5–6 min intervals.
Mice were anesthetized using sodium pentobarbital (60 mg/kg, i.p. injection) and perfused intracardially with 0.9 % saline followed by 4 % formaldehyde. The spinal cord of L4–L5 was removed and post-fixed in 4 % paraformaldehyde for 3 h at room temperature. It was the equilibrated in 30 % sucrose in phosphate buffer overnight at 4 °C. Thirty-micrometer transverse series sections were cut on a cryostat and stored in phosphate buffer. For immunofluorescence staining, free-floating sections were blocked in TBS containing 5 % donkey serum at room temperature for 2 h and then incubated in primary antibody at 4 °C overnight. Sections were then washed in PBS (3 times for 5 min each) followed by incubation in secondary antibody at room temperature for 2 h and additional washing. Sections were mounted on slides and covered with 90 % glycerin and then observed under a confocal microscope (FluoView FV1000; Olympus). The anti-E-cadherin antibody (Millipore) was diluted 1:200.
Western Blotting and Co-immunoprecipitation
Mice were anesthetized using sodium pentobarbital (60 mg/kg i.p.). The dorsal half of the fourth to fifth lumbar spinal cord was removed, immediately frozen in liquid nitrogen, and stored at −80 °C. The tissues were sonicated in ice-cold (4 °C) RIPA lysis buffer (Beyotime Institute of Biotechnology) containing a cocktail of protease and phosphatase inhibitors. After incubation on ice for 15 min, homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C. Protein concentrations were measured using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce). Protein samples were then denatured at 95 °C and separated by 8 % SDS-PAGE. After transfer to PVDF membranes and blocking with 5 % nonfat milk, the membranes were incubated overnight at 4 °C with anti-E-cadherin and anti-GAPDH (both 1:1000; Abcam) primary antibodies. The membranes were washed with wash buffer and incubated for 2 h with alkaline phosphatase-conjugated secondary antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature. The immune complexes were then detected using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate assay kit (Sigma, St. Louis, MO, USA). Western blots were analyzed using densitometry with Adobe Photoshop software (Adobe Systems Inc.).
To detect the p120ctn associated with E-cadherin, the tissue proteins of the spinal cord were obtained for co-immunoprecipitation experiments. Tissues were lysed with co-immunoprecipitation lysis buffer (20 mmol/L Tris-HCl pH 7.5, 137 mmol/L NaCl, 100 mmol/L NaF, 10 % glycerol, 1 % Nonidet P-40, 1 mmol/L PMSF, and protease inhibitor cocktail; Sigma). After brief sonication, lysates were centrifuged at 12,000 rpm at 4 °C. Supernatants were pre-cleared and then incubated with E-cadherin-specific antibody at 4 °C and protein A/G plus agarose beads (Santa Cruz; SC-2003) for 2 h at 4 °C. The beads were pelleted by centrifugation and then washed four times with lysis buffer. After the extensive wash with lysis buffer, the immunoprecipitated proteins were dissolved in 8 % SDS-PAGE and analyzed by Western blotting with p120ctn antibody (1:1000, BD Biosciences).
All data were presented as means ± SEM, and differences were considered significant when P < 0.05. SPSS Rel 15 (SPSS Inc.) was used to perform all statistical analyses. Alterations in protein expression and the behavioral responses to thermal stimuli over time among groups were assessed using one-way and two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni’s post hoc tests, respectively.
CCI Downregulated E-cadherin/p120ctn in the Spinal Dorsal Horn
The PWL of CCI mice decreased on day 3 after CCI and was further reduced on day 7 compared with sham-operated mice (Fig. 1a). This suggests that hyperalgesia had developed. Central or peripheral injury is associated with synaptic structural and plasticity changes in the spinal dorsal horn (Belyantseva and Lewin 1999; Tan et al. 2008). The cadherin family of adhesion proteins has well-described roles in the development, maintenance, and plasticity of synaptic connectivity (Huntley 2002). Therefore, we examined the expression of E-cadherin in CCI mice. Western blotting revealed that CCI resulted in gradually decreased expression of E-cadherin in the spinal cord (Fig. 1b). p120ctn binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which plays a role in regulating cell adhesion (Petrova et al. 2012). Therefore, we next investigated whether p120ctn is associated with the changes in the levels of E-cadherin. We chose the two most obvious points of behavior on days 7 and 14 and analyzed p120ctn by Western blotting. Data revealed that the total p120ctn remained unchanging (Fig. 1c), whereas membrane-associated p120ctn-E-cadherin was significantly decreased after the operation (Fig. 1d). These results suggest that CCI resulted in the gradual decrease of both E-cadherin and membrane-associated p120ctn, whereas total p120ctn levels were unchanged.
GDNF Recruited Intracellular p120ctn to the Juxtamembrane Domain of E-cadherin
The above data revealed that E-cadherin-associated p120ctn decreased significantly after CCI. p120ctn is required for E-cadherin-induced cell adhesion; this association regulates a key event that is necessary for the transition from loose to tight adhesion, and is potentially required for compaction. Next, we assessed whether the loss of E-cadherin-associated p120ctn after CCI could be reversed by treatment with GDNF. The animals were treated with GDNF or PBS daily for 14 days post-operation and then sacrificed. Examination of the lumbar spinal cords of GDNF-treated animals revealed that E-cadherin-associated p120ctn levels were normal and were indistinguishable from sham control animals. In contrast, E-cadherin-associated p120ctn levels in mice exposed to PBS were diminished, as expected (Fig. 2). Because E-cadherin-associated p120ctn levels were significantly diminished 7 days after the operative procedure (Fig. 1d), the restoration of E-cadherin-associated p120ctn by GDNF treatment suggests that the CCI-induced loss of E-cadherin-associated p120ctn is reversible.
GDNF Attenuates Thermal Hyperalgesia and Restores E-cadherin Expression in a Mouse Model of CCI
Previous results revealed that treatment with GDNF could reverse both abnormal pain sensation and the loss of synaptic E-cadherin in L5 spinal nerve transaction model (Patil et al. 2011). Therefore, we assessed whether GDNF exerts similar effects in an experimental model of CCI in mice. Intrathecal GDNF or PBS was administered continuously for 14 days after the operation. Mice with PBS-treated CCI exhibited significant hyperalgesia compared with sham control, whereas thermal hyperalgesia was reduced by GDNF treatment compared with PBS (Fig. 3a). Next, we determined whether GDNF could reverse the effects of CCI on decreased E-cadherin expression. In PBS-treated animals, E-cadherin levels were reduced significantly compared with sham control. In contrast, E-cadherin levels were similar to sham control in animals co-treated with GDNF (Fig. 3b, c). Therefore, these data suggest that intrathecal GDNF could reverse both abnormal pain sensation and the loss of synaptic E-cadherin expression in a CCI-induced neuropathic pain model.
The Effect of DECMA-1 on GDNF-Induced Analgesia and the Recruitment of p120ctn
We next assessed the potential causal involvement of E-cadherin in the analgesic effects of GDNF on CCI-induced hyperalgesia. E-cadherin was inhibited using DECMA-1, which targets the extracellular domain of E-cadherin both in vitro and in vivo (Brouxhon et al. 2013). Intact or CCI mice were first treated with DECMA-1 or IgG to investigate the effect of DECMA-1 on the nociceptive response. There was no change in hyperalgesia in the ipsilateral paw to thermal stimuli in mice treated with either DECMA-1 or IgG (Fig. 4a). Next, we administered DECMA-1 or IgG intrathecally from day 1 after surgery, 30 min prior to GDNF administration. Treatment with the DECMA-1 significantly decreased the analgesic effects of GDNF compared with IgG control mice (Fig. 4b). Therefore, these results suggest that E-cadherin plays an essential role in GDNF-induced analgesia; as such, blocking E-cadherin might antagonize the effects of GDNF on relieving neuropathic pain.
Next, we investigated whether E-cadherin is also involved in the GDNF-induced increase in E-cadherin-associated p120ctn. Co-immunoprecipitation analysis revealed that prior treatment with DECMA-1 inhibited the effect of GDNF on E-cadherin-associated p120ctn (Fig. 4c). Therefore, the data suggest that E-cadherin plays a role in the effect of GDNF on the increase in E-cadherin associated p120ctn.
This study revealed that E-cadherin/p120ctn signaling mediates the analgesic effects of GDNF during neuropathic pain. There were three principle findings: (1) Nerve injury (CCI treatment) caused a rapid loss of E-cadherin expression. Membrane-associated p120ctn, which can positively modulate the adhesion of E-cadherin, also decreased rapidly. (2) GDNF could recruit p120ctn from the cytoplasm to the juxtamembrane domain. Previous work revealed that E-cadherin/p120ctn signaling is important for axonal outgrowth and regulates adhesion. Therefore, the E-cadherin/p120ctn signaling restored by GDNF plays roles in pathophysiological conditions such as chronic pain. (3) Blocking E-cadherin signaling using a functional blocking antibody inhibited the protective effects of GDNF. Taken together, these findings suggest that E-cadherin/p120ctn signaling might be involved in the analgesic effects of GDNF in CCI.
The cell adhesion molecule E-cadherin is important for the formation, maintenance, and regulation of adherens junctions in the epithelial tissues (Shapiro et al. 1995). In addition to its roles in cell adhesion, research increasingly suggests that E-cadherin might be crucial for synapse formation and maintenance (Huntley 2002). For example, the conditional knockout of E-cadherin in individually cultured cortical neurons resulted in a selective reduction in the number of dendritic GABAergic synapses (Fiederling et al. 2011). Furthermore, E-cadherin signaling might be involved in cooperative interactions with other synaptic adhesion molecules (Stan et al. 2010) such as neuroligin-2, which is thought to be crucial for the formation and plasticity of GABAergic synapses (Poulopoulos et al. 2009). In the brain, E-cadherin plays roles in synapse formation and plasticity. However, its localization and roles in injury-related plasticity have not been explored thoroughly in the adult spinal cord, although E-cadherin was distributed in lamina II (Patil et al. 2011). As a consequence of peripheral nociceptor hyperactivity, dramatic secondary changes in the spinal cord dorsal horn also occur in lamina II. Moreover, peripheral nerve crush or axotomy is associated with synaptic structural plasticity within the neuropil of lamina II. Therefore, E-cadherin might regulate synaptic structural plasticity within the spinal cord dorsal horn in response to nerve injury. Previous studies demonstrated that the expression of E-cadherin disappeared by day 7 after axotomy and reappeared following nerve ligature (partial axonal regeneration model) on day 63. In contrast, it remained undetectable following nerve clipping (complete degeneration model) (Seto et al. 1997). Consistent with this, our results also revealed a rapid-onset decreased expression of E-cadherin in the spinal dorsal horn after CCI.
Recent work has begun to elucidate a pivotal role for cadherins and catenins in synaptic development. These proteins not only function as adhesive scaffolds but also help orchestrate synaptic assembly and the subsequent synaptic plasticity (Chen et al. 2012; Marrs et al. 2009). p120ctn is a catenin that binds directly to the juxtamembrane domain of E-cadherin, suggesting a role in regulating cell–cell adhesion (Pieters et al. 2012). In aggregation assays, p120-uncoupled E-cadherin failed to mediate tight adhesion, suggesting that p120ctn is required for the E-cadherin-regulated transition from loose to tight adhesion (Thoreson et al. 2000). In the present study, we found that membrane-associated p120ctn was also decreased significantly after CCI, whereas total p120ctn remained unchanged. Previous work reported that p120ctn exists in two pools: one fraction being bound to cadherins and a second fraction existing as a soluble cytoplasmic pool (Noren et al. 2000). Under normal situations, the cytoplasmic pool of p120ctn is small (Thoreson et al. 2000). Our results predict that the loss of E-cadherin will increase the pool of p120ctn; this will in turn stimulate increased Cdc42 and Rac1 activity and decreased RhoA activity. This results in increase of the migratory activity of the cells. Therefore, during neuropathic pain, cell–cell adhesion in the spinal dorsal horn shifts the balance of p120ctn from the cadherin-bound pool to the cytoplasmic pool.
It is thought that the analgesic properties of GDNF reflect broad neuroprotective effects on injured neurons (Cheng et al. 2002; Pascual et al. 2011). A recent study presented evidence that the activation of GDNF signaling via NCAM has the potential to ameliorate neuropathic pain (Sakai et al. 2008). This raises the possibility that intrathecal GDNF might proactively signal through NCAM to maintain or rebuild the synaptic structural and/or molecular architecture altered by nerve injury. Because NCAM and cadherins belong to a family of cell adhesion molecules, cadherins might orchestrate such molecular changes because they are one of the first molecules recruited to synaptic contacts (Jontes et al. 2004). In the present study, GDNF, which relieved CCI-induced pain, could modulate E-cadherin expression; however, E-cadherin signaling alone was not required for the development of neuropathic pain (Fig. 4a). This is in marked contrast to our finding that an E-cadherin blocking antibody inhibited the effect of GDNF. Therefore, we speculate that E-cadherin signaling alone is not required for the development of neuropathic pain but might be involved in the analgesic effects of GDNF.
In conclusion, this study revealed that GDNF attenuated thermal hyperalgesia in an experimental mouse model of CCI and that E-cadherin/p120ctn signaling might play a role in the effect of GDNF on attenuating heat hyperalgesia. However, our study assessing the analgesic mechanism of GDNF mainly focused on the cytomembrane expression of E-cadherin/p120ctn; therefore, the intercellular signal transduction pathway has not been established. For example, it is unclear whether PI3K/Akt/mTOR signaling downstream from E-cadherin mediates the effects of GDNF (Brouxhon et al. 2013). Therefore, other mechanisms might be involved in the effects of GDNF. In the present study, we revealed that E-cadherin/p120ctn signaling mediated the analgesic effects of GDNF for the first time. We hope that these findings might help our understanding the analgesic mechanisms of GDNF and represent a novel strategy for relieving neuropathic pain.
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The study was supported by grants from National Nature Science Foundation of China (30901402、30900417) and the Educational Department Science Research Foundation of Jiangsu Province (08KJB180011 and 09KJD320008).
Conflict of Interest
The authors declare that there are no conflicts of interest.
Cunjin Wang, Hongjun Wang, and Jun Pang contributed equally to this work.
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Wang, C., Wang, H., Pang, J. et al. Glial Cell-Derived Neurotrophic Factor Attenuates Neuropathic Pain in a Mouse Model of Chronic Constriction Injury: Possible Involvement of E-cadherin/p120ctn Signaling. J Mol Neurosci 54, 156–163 (2014). https://doi.org/10.1007/s12031-014-0266-y
- Glial cell-derived neurotrophic factor
- p120 catenin
- Neuropathic pain
- Chronic constrictive injury
- Spinal cord