Riluzole Promotes Neurite Growth in Rats after Spinal Cord Injury through the GSK-3β/CRMP-2 Pathway

Abstract

Spinal cord injury (SCI) is a disastrous event that often leads to permanent neurological deficits involving motor, sensory, and autonomic dysfunctions in patients. Accumulating research has demonstrated that riluzole may play crucial roles in the process of spinal tissue repair, but the underlying mechanisms remain elusive. This study verified the effectiveness of riluzole and speculated that a riluzole-afforded protection mechanism may be associated with the glycogen synthase kinase-3 beta (GSK-3β)/collapsin response mediator protein-2 (CRMP-2) pathway in rats after spinal cord injury. Here, a modified Allen’s weight dropping model was generated and riluzole at 4 mg/kg was injected intraperitoneally after surgery and twice a day for 7 consecutive days. At 6 weeks after SCI, we found that riluzole treatment reduced the central cavity size of the spinal cord and improved neurological functions. Meanwhile, riluzole-treated rats exhibited shorter latency and larger amplitude in motor evoked potentials and somatosensory evoked potentials, compared with vehicle-treated rats. Furthermore, Western blotting and immunofluorescence data revealed that the expression levels of GSK-3β and phosphorylated-GSK-3β were lower in riluzole-treated SCI rats compared with vehicle-treated rats. We next detected the expression CRMP-2 and phosphorylated CRMP-2 and found that the expression of CRMP-2 showed no difference between the riluzole-treated and vehicle-treated groups; however, administration of riluzole downregulated phosphorylated CRMP-2 expression. The current findings suggest that after SCI, administration of riluzole promotes neurological functional restoration, which may be associated, in part, with its activation of the GSK-3β/CRMP-2 signaling pathway.

INTRODUCTION

Spinal cord injury (SCI) is a disastrous event that often leads to permanent neurological deficit involving motor, sensory, and autonomic dysfunctions in patients. Millions of people suffer from SCI-related neurological complications, which put a huge burden on the society and economy.¹⁾ Although there is currently no cure for SCI, accumulating research has demonstrated that axon regeneration plays a crucial role in neural function restoration after SCI.^2,3) As such, current therapeutic strategies to increase intrinsic axon regeneration to promote functional outcomes after SCI have attracted tremendous attention.

In the past several decades, researchers have begun to dissect the differences in the regeneration capacity of different models; however, a breakthrough in the regeneration of central nervous system (CNS) nerve axons remains elusive.^4–6) Seminal studies demonstrated that CNS neurons with severely damaged axons were able to regenerate in adult rats when autologous sciatic nerve segments were transplanted into the spinal cord.^7,8) This important finding shows that mature neurons retain a certain regenerative ability. Furthermore, glycogen synthase kinase-3 beta/collapsin response mediator protein-2 (GSK-3β/CRMP-2) proved to be the key mediator of signaling networks in axon regeneration.^3,9,10) GSK-3β is as a serine/threonine kinase, and serine phosphorylation at position 9 of GSK-3β inhibits its activities. In neurons, GSK-3β has been shown to mediate microtubule stabilization that regulates axon growth.⁹⁾ This stabilization of microtubules is achieved by adjusting the downstream target of GSK-3β, CRMP-2.¹¹⁾ CRMP-2, in turn, plays a key role in axon growth and neuronal differentiation, and is highly upregulated during early neuronal development. However, phosphorylation of CRMP-2 (p-CRMP-2) by GSK-3β reduces its binding capacity to tubulin, resulting in restricted axon growth.⁴⁾

Riluzole, a sodium channel blocker and inhibitor of glutamatergic neurotransmission, has been widely used in preclinical studies of acute and chronic neurological diseases, including stroke, spinal cord injury, Alzheimer’s disease, Parkinson’s disease, and depression.^12,13) With satisfactory results, riluzole has been approved as a conventional treatment for amyotrophic lateral sclerosis (ALS). It is noteworthy that the phase IIB/III clinical trials to evaluate the effectiveness and safety of riluzole in patients with cervical SCI, which began in 2014, will be finished in 2021.¹⁴⁾ Previous evidence indicated that the neuroprotective effect of riluzole may be achieved by promoting axon repair in the corticospinal tract after SCI, but the underlying molecular mechanisms remain unclear.¹⁵⁾ However, emerging evidence in preclinical studies suggests that riluzole regulates the expression of GSK-3β in the treatment of ALS, Alzheimer’s disease, and melanoma.^16,17) Therefore, we hypothesized that riluzole may also modulate the expression of GSK-3β after SCI, to promote axon repair by acting on its downstream receptor, axon-related CRMP-2.

Our previous studies have verified that riluzole affects the expression of axons marker(s) after SCI.¹⁸⁾ The present study was designed to demonstrate that activation of the GSK-3β/CRMP-2 signaling pathway plays a protective role after SCI.

MATERIALS AND METHODS

Ethics Statement

Adult female (weight, 230 ± 10 g) Wistar rats were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animal maintenance was performed in accordance with the guidelines published by the Chinese National Institutes of Health. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, China).

Animal Grouping and SCI Procedure

Seventy-two adult female rats were assigned randomly into four groups, including control, sham, vehicle, and riluzole (n = 18 each). The vehicle and riluzole groups underwent surgery and weight-dropping, which caused SCI. Rats in the sham group only underwent surgery without weight-dropping. Rats in the control group were normal rats without any surgical intervention or weight-dropping.

The SCI procedure was performed according to our previous study.¹⁹⁾ Briefly, rats undergoing surgery were anesthetized using a mixture of oxygen and isoflurane. The surgical area located on the back was shaved and disinfected with iodophor. A midline incision was made and the lamina of T10 was exposed. Laminotomy of T10 was performed and the spinal cord was clearly exposed. Next, an IMPACTOR MODEL III (State University of New Jersey, U.S.A.) was applied for the weight-dropping experiment. The rod parameters were set to a height of 25 mm, weight of 10 g, and diameter of 3 mm. The incision was then closed with sterile sutures, after which the rats were wrapped in an electric blanket for recovery.

Animal Care and Drug Administration

Rats were individually housed in a cage and food and water were provided as necessary. Postoperatively, the rats’ bladders were evacuated four times daily for 7 d to prevent urinary tract infections and postoperative bladder filling. The perineum and hindquarters were cleaned with salt water daily and then dried. Animals were intraperitoneally injected with vehicle or riluzole at 4 mg/kg immediately after surgery and then twice a day for 7 consecutive days.¹⁸⁾

Behavior Assay

The extent of locomotion recovery in animals was evaluated with the Basso, Beattie, and Bresnahan (BBB) score²⁰⁾ and inclined plane test.²¹⁾ BBB scores were used to assess paw placement, hind limb movement, gait, and coordination, and the scores ranged from 21 (normal) to 0 (paralysis). The inclined plane test was used to assess strength of the hind paw. Briefly, the rats were placed on a flat plate that initially had an angle of 0° but continued to increase gradually. The maximum angle that the rat remained on the plate for 5 s was then recorded and the average of three measurements was taken. Evaluation of the inclined plane test and BBB scores was performed at 1, 7, 14, 21, 28, 35, and 42 d after SCI.

Electrophysiological Evaluation

Motor evoked potentials (MEPs) and somatosensory evoked potentials (SEPs) were recorded to assess the recovery of the motor and sensory systems after SCI. The MEP and SEP assays were performed as previously described.²²⁾ Briefly, rats were anesthetized with a mixture of oxygen and isoflurane. Stimulator electrodes were then inserted subcutaneously above the anterior fontanelle and the active recording electrodes were placed in each achilleas tendon to record MEPs. Meanwhile, stimulator electrodes were inserted in the skeletal muscles of both hind limbs and the active recording electrodes were placed subcutaneously in the bilateral sensorimotor cortex to record SEPs. The N1-P1 amplitude and N1 peak latency were used to analyze the effect of neurological recovery.

Histology and Immunofluorescence

All rats were anesthetized using a mixture of oxygen and isoflurane at day 42 after SCI. They were sequentially perfused transcardially with 0.9% saline for 2 min, fixed with 4% paraformaldehyde for 12 h, and dehydrated with 30% sucrose for 72 h. Spinal cord segments, including the injury site, were dissected out and then longitudinal sections (20 µm) were cut (CM1950, Leica, Wetzlar, Germany) for subsequent immunofluorescence and histology. The spinal cord tissue containing 0.5 cm of the injury center was excised and 30 sections of each spinal cord were generated and numbered 1–30. The same numbers were then used in the different groups for comparison and statistical analyses. For immunofluorescence, the sections (20 µm) were incubated with primary antibodies including NF200, MBP, and p-GSK-3β overnight at 4 °C. After phosphate-buffered saline washes, sections were incubated with donkey anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG for 30 min at 37 °C. Sections were washed and incubated with 4′,6-diamidino-2-phenylindole for 30 s. A laser scanning confocal microscope (Nikon, Tokyo, Japan) was used to capture images. Image J was used to evaluate fluorescence intensity of the NF200 and MBP. The number of positive cells in each slice was recorded for the analysis of expression of p-GSK-3β. Hematoxylin and eosin (H&E) staining was performed according to the manufacturer’s protocol (C0105, Beyotime, China), and the cavitation area was gauged by histopathologists using a laser scanning confocal microscopy (Nikon). The information and dilution factors of the antibodies are summarized in Table 1.

Table 1. Antibody Information Sheet

Antibody	Catalogue #	Concentration	Isotype control
NF200	Sigma, N5389-.2ML, mouse	IF 1 : 50	Mouse IgG
MBP	Millipore, AB5864, Rabbit	IF 1 : 200	Rabbit IgG
GAP-43	CST, # 8945, Rabbit	WB 1 : 1000	Rabbit IgG
GSK-3β	CST, #12456, Rabbit	WB 1 : 1000	Rabbit IgG
CRMP-2	CST, #9393, Rabbit	WB 1 : 1000	Rabbit IgG
p-GSK-3β	CST, #5558, Rabbit	WB 1 : 1000	Rabbit IgG
p-CRMP-2	CST, #9397, Rabbit	WB 1 : 1000	Rabbit IgG
Goat anti-mouse IgG	Invitrogen, A32723	IF 1 : 100 WB 1 : 5000	n/a
Donkey anti-rabbit IgG	Invitrogen, A32754	IF 1 : 100 WB 1 : 5000	n/a

Western Blot

Western blot was utilized to evaluate Gap-43, p-GSK-3β, GSK-3β, p-CRMP-2, and CRMP-2 expression. Total protein was extracted and then quantitatively analyzed using the bicinchoninic acid method. A 10% separation gel and 5% resolving gel were the prepared for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and proteins subsequently transferred onto a nitrocellulose membrane (Bio-Rad, CA, U.S.A.). Fat-free milk (5%) was used to block the membranes for 1 h, after which they were incubated with primary antibodies including rabbit-anti-Gap-43, rabbit-anti-GSK-3β, rabbit-anti-CRMP-2, p-GSK-3β, and p-CRMP-2 overnight at 4 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, U.K.) was used as an internal control. Then, the membranes were incubated with secondary antibody for 1 h at 37 °C. The ECL detection system (Bio-Rad) was then used to visualize protein bands, and Image J was used to analyze the intensities.

Statistical Analysis

Data analysis was performed using the SPSS 23.0 software (IBM Corporation, NY, U.S.A.). Comparisons between two groups were analyzed using the Student’s t test. Differences across multiple groups were assessed by ANOVA, followed by Tukey’s post hoc analysis. Two-way mixed model ANOVA was used to analyze behaviors including the inclined plane test and BBB scores, followed by Tukey’s post hoc analysis. Statistical significance was considered as p < 0.05.

RESULTS

After SCI Administration of Riluzole Reduced the Central Cavity and Improves Behavioral Scores

The central cavity size was significantly smaller in the riluzole group (Figs. 1c, d) compared with the vehicle group, as measured by H&E staining. Neurofunctional deficits were assessed using BBB scores and the inclined plane test after SCI. As shown (Fig. 1e), riluzole-treated rats exhibited higher BBB scores at 6 weeks after SCI, compared with vehicle-treated rats. Similarly, riluzole-treated rats were able to stay on the plate at increased higher angles in the inclined plane test compared with vehicle-treated rats (Fig. 1f). These results indicate that administration of riluzole reduced the central cavity and improved behavioral functions after SCI.

Fig. 1. Riluzole Reduced the Central Cavity and Improved Behavioral Scores

(a) Experimental protocol. (b) Molecular formula of riluzole. (c) Representative H&E-staining of the spinal cord at 6 weeks after SCI. (Scale bar: 1000 µm.) (d) Quantification of the lesion area between vehicle group (n = 6) and riluzole group (n = 6). p < 0.05. (e–f) Quantification of BBB scores and the inclined plane test in the four groups (control, sham, vehicle, and riluzole): n = 18/group, p < 0.001 (***): riluzole group versus vehicle group.

Treatment with Riluzole Accelerated the Evoked Potential Restoration in Rats after SCI

Evoked potential conductivity was measured at 6 weeks after SCI. In the SMPs, different amplitude and latency scales were found among the control, sham, vehicle, and riluzole groups (Figs. 2a–d). The N1 peak latency (the first positive deflection) in the riluzole group was significantly shortened compared with that in the vehicle group (Fig. 2i). In addition, riluzole-treated rats had a larger N1-P1 amplitude than rats in the vehicle group (Fig. 2j). SEP waves in the four groups showed obvious waveform variation (Figs. 2e–h). Rats in the riluzole group had shorter N1 peak latency compared with rates in the vehicle group (Fig. 2k). Meanwhile, the N1-P1 amplitude was significantly different between rats in the riluzole and vehicle groups (Fig. 2l). These results indicate that riluzole treatment improved evoked potential conductivity after SCI.

Fig. 2. Treatment with Riluzole Accelerated the Evoked Potential Restoration in Rats after SCI

(a–d) Representative waveform of motor evoked potentials (MEPs). (e–h) Representative waveform of somatosensory evoked potentials (SEPs). (i, j) Quantification of the latency and amplitude of MEPs. (k, l) Quantification of the latency and amplitude of SEPs: n = 6/group, NS: control group versus sham group; p < 0.001 (***): vehicle group versus sham group; p < 0.05 (#), p < 0.01 (##): riluzole group versus vehicle group.

Riluzole Enhanced the Expression of NF200 and MBP in Rats after SCI

To determine the effect of riluzole on neurofilaments protein and myelin, we detected the expression of NF200 and MBP, respectively, by immunofluorescence. As shown in Fig. 3a, rats in the riluzole group exhibited a higher integrated optical density of NF200 compared with rats in the vehicle group (Fig. 3b). Meanwhile, the integrated optical density of MBP was significantly different between rats in the riluzole and vehicle groups (Fig. 3c). In addition, Western blot was utilized to evaluate Gap-43. As shown in Fig. 3d, expression of Gap-43 was decreased in the vehicle group compared with the control and sham groups, whereas riluzole treatment rescued its expression. Collectively, the results indicate that riluzole enhanced expression of NF200 and MBP in rats after SCI.

Fig. 3. Riluzole Enhances Expression of Neurofilaments and Myelin in Rats after SCI

(a) Representative immunofluorescence image of spinal cord double-stained for NF200 (green) and MBP (red) at 6 weeks after SCI. Scale bar: 50 µm. (b, c) Quantitative analysis of the expression levels NF200 and MBP in the four groups. (d) Western blot analysis of Gap-43 protein expression in the four groups at 6 weeks after SCI: n = 6/group, NS: control group versus sham group; p < 0.001 (***): vehicle group versus sham group; p < 0.05 (#): riluzole group versus vehicle group.

Riluzole Activated the Axon-Related GSK-3β/CRMP-2 Signaling Pathway after SCI

The GSK-3β/CRMP-2 signaling pathway plays a key role in axonal regeneration.⁹⁾ CRMP-2 is related to axon growth and neuronal differentiation; however, GSK-3β phosphorylates CRMP-2 and reduces its binding capacity to tubulin, resulting in restricted axon growth.⁴⁾ To further identify the underlying mechanism of riluzole mediated axonal growth, we measured the levels of GSK-3β, p-GSK-3β, CRMP-2, and p-CRMP-2 using Western blot. As shown in Fig. 4a, the expression of GSK-3β and p-GSK-3β was decreased in the riluzole group compared with the vehicle group (Figs. 4d, e). Although the expression of CRMP-2 was not significantly different between the riluzole and vehicle groups, administration of riluzole downregulated the expression of p-CRMP-2 (Figs. 4b, f, g). Meanwhile, immunofluorescence was utilized to further detect the expression of p-GSK-3β. As shown in Fig. 4c, riluzole treatment lowered the expression of p-GSK-3β compared with vehicle-treated rats (Fig. 4h). These results indicate that riluzole treatment activated the axon-related GSK-3β/CRMP-2 signaling pathway after SCI.

Fig. 4. Riluzole Activates the Axon-Related GSK-3β/CRMP-2 Signaling Pathway after SCI

(a, d, e) Representative Western blots and quantification of p-GSK-3β and GSK-3β in the spinal cord at 6 weeks after SCI. (b, f, g) Representative Western blots and quantification of p-CRMP-2 and CRMP-2 in the spinal cord at 6 weeks after SCI. (c) Representative immunofluorescence image p-GSK-3β (red) in the spinal cord at 6 weeks after SCI (scale bar: 50 µm). (h) Quantitative analysis of p-GSK-3β expression in the four groups: n = 6/group, NS: control group versus sham group; NS, p < 0.05 (*), p < 0.001 (***): vehicle group versus sham group; p < 0.05 (#): riluzole group versus vehicle group.

DISCUSSION

Riluzole, a sodium channel blocker and inhibitor of glutamatergic neurotransmission, has been approved by the U.S. Food and Drug Administration for the treatment of ALS. Because riluzole is transported efficiently across the blood brain barrier and possesses superior bioavailability, it is widely used in preclinical studies of acute and chronic neurological diseases.²³⁾ Accumulating evidence has demonstrated that riluzole may be a promising therapeutic candidate for SCI, via alleviation of oxidative stress, reduction of glutamate-mediated excitotoxicity, inhibition of inflammatory responses, and promotion of neurotrophin expression.^24–26) In our study, a modified Allen’s weight dropping model was used to study the neuroprotective effect of riluzole. Our results showed that administration of riluzole reduced the central cavity, accelerated the evoked potential conductivity, and expedited the restoration of neurological function at 6 weeks after SCI. Additionally, we observed riluzole-treated rats exhibited higher expression of NF200 and Gap-43. These findings suggest an increase in axon-related proteins, which may be associated with restoration of evoked potential conductivity and neurological function. In the current study, we assessed the effect of riluzole on evoked potential and axon regeneration after SCI, and speculated that the GSK-3β/CRMP-2 signaling pathway may be involved in the underlying mechanism.

The primary function of the axon is to transmit excitatory impulses generated by the neuronal cell body to other neurons or their effectors.²⁷⁾ Axonal rupture, as a result of SCI, affects the transmission of these excitatory impulses and manifests as muscle atrophy and loss of the corresponding tendon reflex.²⁸⁾ During the second week after SCI, astrocytes then become hypertrophic and proliferate to form glial scars, which form a physical and chemical barrier against axonal regeneration.^29,30) Therefore, early intervention focusing on axons is an attractive goal. Here, our team applied riluzole in the early stage of SCI, and used MEPs and SEPs to evaluate axon conduction function and detect the expression of axonal markers.

MEPs and SEPs are widely used to evaluate the recovery of the motor and sensory systems after SCI. Evoked potential waveforms convey physiological information, reflecting sequential neurapraxia along the pathway between stimulator electrodes and recording electrodes.^31,32) The potential completely disappeared, the possibility of complete spinal cord injury is very high^31–33); The prolonged latency and decreased amplitude could reflect the dysfunction of nerve conduction function.^31–33) MEPs and SEPs are the objective evaluation of the prognosis of SCI.^31–33) Here, we assessed the conveyed physiological information after SCI and then again after subsequent riluzole treatment. We found that riluzole-treated rats exhibited shorter latency and larger amplitude in motor evoked potentials and somatosensory evoked potentials, compared with vehicle-treated rats. The results showed that riluzole treatment improved the recovery of the motor and sensory systems after SCI.

The integrity of axons is inseparable from the conduction of evoked potentials.^22,32) Gap-43 is regarded as a neurofilament-related protein, which is associated with neurite regeneration after CNS injury.^34,35) The expression of Gap-43 was found to be up-regulated in regenerating and developing neurons.³⁵⁾ Meanwhile, previous evidence showed that Gap-43 also induced neurotransmitter release and enhanced synaptic plasticity.³⁵⁾ Additionally, expression of NF200 is also used to evaluate axon regeneration. Our study showed that after SCI, administration of riluzole increased the expression of both Gap-43 and NF-200. Thus, we speculate that riluzole promoted the regeneration of axons after SCI.

Here, we observed that the GSK-3β/CRMP-2 signaling pathway may be involved in the riluzole-afforded protection mechanism after SCI. The role of GSK-3β in the CNS is multifaceted, and includes regulation of neurofilament growth, participation in immune responses, and involvement in the cell cycle and apoptosis.^4,9,17) The pharmacological inhibition of GSK-3β leads to microtubule polymerization and extensive axon growth.^4,36) Serine phosphorylation at position 9 of GSK-3β inhibits its activities. Phosphorylation at the Ser9 site of GSK-3β could generate a pseudo-substrate that binds to the catalytic structure of GSK3, thereby preventing the substrate from binding to the active site of the enzyme and inhibiting GSK-3β activity.³⁷⁾ In this study, riluzole-treated rats exhibited lower expression of GSK-3β and p-GSK-3β compared with vehicle-treated rats, suggesting that riluzole may reduce GSK-3β activity in rats after SCI. The effect of GSK-3β on neurofilament growth is known to be achieved through its downstream activation of CRMP-2. Phosphorylation of CRMP-2 by GSK-3β reduces its binding capacity to tubulin, resulting in restricted axon growth.⁴⁾ The phosphorylation of CRMP-2 at Thr-514 may prevent the copolymerization of CRMP-2 with tubulin dimers into microtubules until reaching the growth cone, or it may induce the dissociation of CRMP-2 from microtubules, thus affecting axon growth and function.³⁸⁾ We therefore detected the expression CRMP-2 and p-CRMP-2 and found that the expression of CRMP-2 showed no significant difference between riluzole-treated and vehicle-treated rats. However, administration of riluzole downregulated p-CRMP-2 expression. These findings suggest that the riluzole-afforded protection mechanism after SCI may be associated, in part, with its activation of the GSK-3β/CRMP-2 signaling pathway. Furthermore, we speculate that riluzole affects GSK-3β by inhibiting glutamate. James et al. found that glutamate blockade with riluzole inhibited signaling through the phosphatidylinositol 3-kinase/AKT pathways, an important upstream pathway of GSK-3β.³⁹⁾ In addition, Vallée et al.¹⁶⁾ reported that the intervention with riluzole may regulate GSK-3β-mediated targeting of the WNT/β-catenin pathway in Alzheimer’s disease. However, further research is needed to verify the above effects of riluzole in SCI.

In summary, our current study showed that riluzole reduced the central cavity and improved functional recovery in rats after SCI. The underlying mechanism may, in part, be associated with activation of the GSK-3β/CRMP-2 signaling pathway in the spinal cord. Thus, riluzole may be a promising therapeutic candidate for SCI.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81901241).

Conflict of Interest

The authors declare no conflict of interest.

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