Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Abstract

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.

Introduction

Neuropathic pain is defined as the pain initiated or caused by a primary lesion or dysfunction in the nervous system (IASP). Damage to peripheral nerves may produce hyperalgesia or allodynia (Devor 1994; Song et al. 2003). These symptoms are associated with changes in neurotransmitter phenotype (Hains et al. 2004), synaptic plasticity and excitability of primary afferent nociceptors and dorsal horn neurons (Woolf 2007; Salter 2005). Despite extensive researches into neurobiological mechanisms of neuropathic pain during the past decades, our understanding of this disorder is still incomplete. The formation of specific synapses between pre- and post-synaptic neurons leading to organized neuronal networks may offer an explanation for the development of neuropathic pain (Chen et al. 2007).

β-Catenin, a peripheral cytoplasmic protein associated with the cytoplasmic domains of cadherins, which is expressed in neurons and localized to synaptic contacts, is involved in synapse formation and neural circuit assembly during development (Kwiatkowski et al. 2007). In the presynapse, β-catenin associated with PDZ-domain containing proteins is required for synaptic vesicle clustering (Bamji et al. 2003; Ziv and Garner 2004). In the postsynapse, neuronal activity modifies spine shape and size and promotes β-catenin localization into spines, which may provide a morphological basis for synaptic plasticity (Kennedy et al. 2005; Segal 2005). The transcription factor menin is a product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene (Chandrasekharappa et al. 1997) and also a critical mediator of synapse formation between central neurons. Postsynaptic expression of menin is necessary for the proper formation of various types of central synapses, both excitatory and inhibitory. Antisense knock-down of menin either prevents synapse formation or significantly reduces synaptic efficacy (Van Kesteren et al. 2001).

In spite of studies that correlate β-catenin and menin in the nervous system, little research is performed into the role of β-catenin and menin in neuropathic pain. This study analyzed spinal cord β-catenin and menin expression in rats subjected to a spared nerve injury.

Materials and Methods

Animals

Thirty-eight adult male Sprague–Dawley rats (220–250 g) obtained from Shanghai Laboratory Animal Center (SLAC) were used in this study. They were housed at room temperature (22–25°C) with a 12 h light/dark cycle. Food and water were available ad libitum. All procedures were carried out with the approval of the Animal Care and Use Committee of Institute of Neuroscience, Shanghai and were consistent with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. All efforts were made to minimize the number of animals used and their suffering. Rats were given 5 days to acclimatize to the vivarium before experimentation. Mechanical withdrawal thresholds and foot deformation were measured before the SNI surgery and on post-SNI surgery days 1, 3, 7, 14 and 21.

Surgical Operation

The SNI models (n = 27) were performed according to the method of Decosterd and Woolf (2000). Under general anesthesia with i.p. sodium pentothal (50 mg/kg) and sterile conditions, the left sciatic nerve and its trifurcation were exposed. We carefully separated the three terminal branches of the sciatic nerve (tibial, common peroneal and sural nerves) and tightly ligated the tibial and common peroneal nerves individually with 5.0 silk which were then cut distally. To prevent regeneration, a section of 2–3 mm of each nerve was subsequently removed. The muscle and skin incisions were closed separately using silk sutures. All animals were allowed to fully recover before being returned to their home room. In sham-operated animals (n = 9), the left sciatic nerve was exposed but not transected. For further comparisons a naïve group (n = 2) was included in which the animals did not undergo surgical manipulation.

Evaluation of Mechanical Allodynia

Each animal was placed in a plexiglas chamber on an elevated mesh screen and allowed to acclimate for 30 min before beginning behavioral experiments. Mechanical allodynia was evaluated by application of von Frey hairs (North Coast Medical, Morgan Hill, CA, USA). Each test consisted of applications of a series of von Frey monofilaments (ranging from 0.07 to 26.0 g) to the lateral plantar surface of the tested paw until it withdrew or for a maximum of 10 s. To avoid sensitization during tests, each trial was taken at 5 min intervals. The withdrawal threshold was determined as the filament at which the animal withdrew its paw at least twice in 5 applications.

Assessment of Foot Deformation

The rat was placed on a plate with a neutral temperature (22–23°C) and the foot deformation was scored according to the method of Nakazato-Imasato and Kurebayashi (2009). Briefly, score 0 if the paw is in normal position with fanned toes; score 1 if the toe is ventroflexed; score 2 if the paw is everted so that only the internal edge of the paw touches the floor.

Immunoblotting

Rats were anesthetized with sodium pentobarbital, and lumbar segments of spinal cord were removed and the dorsal horn was dissected for later use. The Western blot samples were homogenized in lysis buffer containing protease inhibitors and then centrifuged at 8000×g for 20 min at 4°C, and the supernatants were kept for later experiments. The protein concentration was determined using Bradford method (Pierce #23209 Rockford, IL, USA). Protein (20 μg) was separated on a 10% SDS-PAGE gel and transferred to PVDF membrane (Millipore Corporation, Bedford, MA 01730, USA). The membranes were blocked with 10% non-fat milk in TBS buffer at room temperature (22–25°C) for 2 h and then incubated with anti-menin (1:1000, Cell Signal), anti-β-catenin (1:1000, Cell Signal) or anti actin (1:50000) diluted in TBS plus 5% BSA overnight at 4°C. The membranes were washed with 0.1% TBST buffer and incubated for 1 h with anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP) (1:2000, Cell Signal) in 5% non-fat milk/TBS. The immunoreactivity was detected with chemiluminescence reagents provided with the ECL kit and exposured using LAS-4000 Luminescent Imaging Analyzer (FUJIFILM Life Science). The densitometric quantification of immunoreactive bands was performed using ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis

Statistical analyses were carried out with Graphpad Prism software (San Diego, CA, USA). All data are presented as the mean ± SEM. The data among groups were analyzed by Student’s t-test analysis with 2-tailed distribution. For all tests, differences were considered significant at the 5% level (P < 0.05).

Results

Time Course of Mechanical Allodynia

The paw withdrawal threshold (PWT) of SNI rats to the application of von Frey monofilaments was illustrated in Fig. 1. No significant changes in PWT were observed in sham-operated animals (n = 6), ranging from 11.0 ± 1.32 to 15.0 ± 0.00 g during the entire observation period. In contrast, the average paw withdrawal latency of the SNI (ipsilateral) paws (n = 6) reduced to 3.23 ± 0.49 g on day 1 PO, to 0.60 ± 0.09 g on day 3 PO and to 0.21 ± 0.06 g on day 7 PO, while that at the uninjured (contralateral) paw was 12.67 ± 1.48 g on day 1 PO, 15.00 ± 0.00 g on day 3 PO and 13.33 ± 1.05 g on day 7 PO (Fig. 1, P < 0.001). The magnitude of SNI-induced mechanical allodynia remained unchanged for 21 days PO.

Fig. 1.

Mechanical allodynia after spared nerve injury (SNI). Paw withdrawal threshold (PWT) in response to von Frey filament stimulation decreased from 15.00 ± 0.00 g before surgery to 0.21 ± 0.06 g 7 days after surgery. This mechanical allodynia persisted for at least 21 days. Sham operated rats did not show any signs of hypersensitivity. ** P < 0.001 in relation to the corresponding contralateral side. Cont contralateral to injured hind paw; Ipsi ipsilateral to injured hind paw

Time Course of Foot Deformation

As shown in Fig. 2, 53.3% SNI rats (n = 15) demonstrated abnormal posture of the affected limb on day 1 PO. The foot deformation in rats with SNI gradually became more serious and all animals demonstrated abnormal foot posture at >2 weeks PO.

Fig. 2.

Time course of the foot deformation in rats after spared nerve injury (SNI) of the sciatic nerve. Data are expressed as percent of rats displaying each of the following 3 categories of foot posture after the SNI surgery (n = 15). Score 0: normal foot, score 1: foot showing ventroflexed toes, and score 2: foot showing everted paw

β-Catenin and Menin Protein in Rat Spinal Cord

In order to observe the time course of expression of β-catenin and menin in the dorsal horn after SNI, the rats were killed on days 1, 3, 7 and 14 PO (n = 3 for each time point). The expression of β-catenin and menin in the dorsal horn were assayed by Western blot (Fig. 3a, b). Using the immune chemiluminescence density of actin as a standard, we quantified the relative protein levels of β-catenin and menin, and all the data were expressed as ratios (Fig. 3c, d). We could not find any significant difference of β-catenin and menin expression between ipsilateral and contralateral from the naïve (n = 2) and sham (n = 3) groups. However, SNI caused a significant (P < 0.05) elevation in β-catenin expression (Fig. 3a, c) on the ipsilateral side at the days 1–3 PO in relation to the contralateral side and sham group. At day 7 PO, SNI at ipsilateral side also exhibited an increase in β-catenin expression, but it was not significant statistically (P > 0.05). Meanwhile, expression of menin in spinal dorsal horn ipsilateral to SNI significantly increased within day 1 after injury and got to the peak value at the day 3 PO, and then decreased to a lower level (Fig. 3b, d). At the day 14 of post surgery, both β-catenin and menin expression in SNI group decreased to the similar level of naïve and sham groups (Fig. 3c, d).

Fig. 3.

Western blot analysis for β-catenin and menin in rat spinal cord dorsal horn. a, b Representative Western blots showing expression of β-catenin and menin in spinal cord horn of naïve, sham, and SNI groups 1, 3,7, and 14 days after surgery. c, d Statistical pooled results of pixel optical density of β-catenin and menin in spinal cord horn of naïve (n = 2), sham (n = 3), and SNI groups 1, 3, 7, and 14 d after surgery (n = 3 for each time point). * P<0.05 in relation to the corresponding contralateral side; ^# P < 0.05 and ^## P < 0.01 compared with naïve and sham groups. Cont contralateral to injured hind paw; Ipsi ipsilateral to injured hind paw

Discussion

In the present study, we observed an increased expression of β-catenin and menin in the dorsal horn during the early stage of SNI induced neuropathic pain. The increases in β-catenin and menin protein were transient and associated with early behavioral signs of pain. These results provide evidence for a novel mechanism involved in neuropathic pain induced by nerve injury.

SNI produced a significant up-regulation of β-catenin and menin proteins in the spinal dorsal horn and induced severe mechanical hyperalgesia. Interestingly, the time course of increased β-catenin and menin expression was highly correlated with the development of mechanical hyperalgesia after SNI. The mechanical hyperalgesia started from day 1 gradually reached a peak at days 3–7 and lasted for 21 days, while the expression of β-catenin also rose during days 1–7. Therefore, we hypothesized that the upregulation of β-catenin and menin may contribute to the development of neuropathic pain. Decosterd and Woolf (2000) proved that a major feature of the spared nerve injury (SNI) was hypersensitivity to normally innocuous mechanical stimuli while heat sensitivity was unchanged. The related symptoms are due to large low-threshold mechanoreceptor myelinated fibers to produce Aβ fiber-mediated pain (Woolf and Salter 2000), and almost all (98%) Aβ-fibers are mechanosensitive (Jänig et al. 2009). Our results consistently demonstrated that SNI induced significant mechanical hypersensitivity on the ipsilateral side (but not on the contralateral side). However, Choua et al. (2002) demonstrated an increased synaptophysin expression (a presynaptic vesicle protein used to identify synaptogenesis) in the rat spinal cord after sciatic CCI which correlated with thermal hyperalgesia but not tactile allodynia. These results may be due to the different mechanisms underlying the development of pain and central plasticity between the two models.

Although our hypothesis sounds reasonable, the possible mechanisms underlying the role of β-catenin and menin in generating neuropathic pain have not yet been evaluated. According to the theory that sprouting of Aβ-fibers from lamina III into II of spinal cord may result in the development of persistent tactile allodynia (Lekan et al. 1996; Woolf et al. 1992), perhaps, β-catenin contributes to the sprouting of Aβ-fibers through promoting the development of Schwann cell (SC) and oligodendrocyte. After injury of peripheral nerves, Schwann cell (SC), the glial cells of the peripheral nervous system (PNS), are in intimate contact with and exhibit a reciprocal interaction with axons (Esper and Loeb 2004; Hoke et al. 2006). β-Catenin is expressed at key stages of Schwann cell (SC) development. In SC monocultures, β-catenin reduction diminished the proliferative response of SCs to the mitogen β1-heregulin, and, in SC-DRG cocultures, β-catenin reduction inhibits axon-contact-dependent SC proliferation (Gess et al. 2008). Oligodendrocytes are the myelinating cells of the vertebrate CNS (Miller 2002). β-Catenin signaling is active during oligodendrocyte development and remyelination in vivo; dysregulation of Wnt/β-catenin signaling in oligodendrocyte precursors results in profound delay of both developmental myelination and remyelination (Fancy et al. 2009). Furthermore, β-catenin interacts with adhesion molecules such as cadherins, regulators of the synaptic remodeling process, to influence synaptic size and strength (Murase et al. 2002; Sun et al. 2009). These researches proved that the role of β-catenin in sprouting of Aβ-fibers from lamina III to lamina II of the dorsal horn and new synapses formation between neurons may underlie the development of neuropathic pain. The MEN1 gene encoding the tumor suppressor protein menin as a critical mediator of synapse formation between central neurons is also necessary for the proper formation of various types of central synapses (Van Kesteren et al. 2001). Menin is up-regulated during synapse formation; antisense knock-down of it can prevent synapse formation between molluscan neurons in primary cell culture (Van Kesteren et al. 2001). Moreover, menin is essential for canonical Wnt/β-catenin signaling in cultured rodent islet tumor cells, overexpression of menin significantly enhances T cell factor (TCF) gene assay reporter activity in response to β-catenin activation (Chen et al. 2008). It has an essential role in Wnt/β-catenin signaling through histone methylation of downstream target gene promoters (Sawicki et al. 2008). Menin is also able to directly interact with β-catenin and carry β-catenin out of the nucleus (Cao et al. 2009). These results imply that menin may regulate the production of neuropathic pain through interacting with Wnt/β-catenin signaling.

Moreover, it is well accepted that activation of NMDA receptors in the spinal dorsal horn plays crucial roles in the development and maintenance of neuropathic pain following nerve injury (Bennett et al. 2000; Bleakman et al. 2006; Chizh and Headley 2005). NMDA receptor antagonists have been shown to reduce allodynia and hyperalgesia in animal models of neuropathic pain (Fundytus 2001). Furthermore, the spread of excitation from lamina III into superficial dorsal horn lamina, which underlie some forms of Aβ-fibre-mediated allodynia, is mediated by glutamatergic synapses (Schoffnegger et al. 2008). In addition, NMDA receptor activation induces the nuclear translocation of a LAPSER1/β-catenin complex (Schmeisser et al. 2009), and in turn β-catenin redistribution induced by depolarization could be completely blocked by NMDAR antagonists (Murase et al. 2002). On the other hand, NMDA-receptor dependent activation of calpain could induce the cleavage of β-catenin at the N terminus, generating stable, truncated forms, which could result in stimulation of TCF-dependent gene transcription and structural modulation of synaptic junctions (Abe and Takeichi 2007). Therefore, the expression of β-catenin appears to be correlated with NMDAR activity during the development of neuropathic pain after peripheral nerve injury.

In conclusion, β-catenin and menin protein expressions increase significantly after nerve injury initially and these changes may participate in the sprouting of Aβ-fibers and new synapse formation that underlies the development of neuropathic pain. Inhibition of β-catenin and menin protein expression after nerve injury may have therapeutic benefits and relieve symptoms of neuropathic pain.