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Damage to the spinal cord (SC) can result in irreversible impairments or complete loss of motor, sensory, and autonomic functions. Riluzole, a sodium channel-blocker and glutamate inhibitor, is in preclinical use for SC injury (SCI), and curcumin is an intracellular calcium inhibitor that attenuates glutamate-induced neurotoxicity. As riluzole and curcumin have different mechanisms to protect against SCI, we aimed to investigate the neuroprotective effects of a combination of riluzole and curcumin in human astrocytes and white matter injury (WMI) model of SCI. Our data show that a combination of riluzole (1 μM) and curcumin (1 μM) was effective in inhibiting hydrogen peroxide (H 2 O 2 )-induced oxidative dress in astrocytes derived from human SC, however, curcumin alone showed a significant inhibition. In addition, our results demonstrated that curcumin alone downregulates the hypoxia-induced expression of HIF-1, GFAP, and NF-H proteins in WMI, whereas riluzole alone and in combination with curcumin remained ineffective in changing the expression of these proteins. Contrarily, after inhibiting Ca 2+ influx with EGTA, riluzole alone and in combination with curcumin significantly downregulated hypoxia-induced expression of GFAP and NF-H. After analysis of caspase 9 and cleaved caspase 9, we observed that curcumin and riluzole both inhibit apoptosis significantly, whereas their combination remains ineffective. Furthermore, we observed that neuroprotective effects of curcumin and riluzole are mediated through Nrf2/HO-1 signaling. In conclusion, our results demonstrate that curcumin and riluzole protect astrocytes from oxidative stress and white matter from hypoxia. However, their combination is not beneficial to reduce hypoxia-induced astrocytosis, axonal damage, and apoptosis. From our results, it is evident that curcumin is more effective in reducing WMI than riluzole., (Published by Elsevier B.V.)

Spinal cord injury (SCI) triggers pronounced inflammatory responses that are accompanied by neuronal disruption and functional deficits. SCI treatment remains an unmet clinical need. Emerging evidence suggests that riluzole may exert a neuroprotective effect due to its anti-inflammatory properties. However, details of the underlying mechanisms remain poorly defined. The polarization of microglial/macrophages has an important role in neuroinflammation. Here, we examined whether riluzole can exert a neuroprotective effect after acute SCI, and whether this effect is associated with changes in microglia/macrophages polarization. Riluzole (4 mg/kg) or vehicle were injected intraperitoneally (i.p.) in female rats immediately following SCI and repeated for 7 consecutive days (b.i.d.). Compared with vehicle treatment, riluzole-treated SCI rats showed significant higher locomotor scores (Basso, Beattie, and Bresnahan score, Inclined Plane test score, n = 18/group). Riluzole-treated rats also developed smaller spinal cavities, showed higher levels of myelin basic protein (MBP) and neurofilament (NF)200 immunoreactivities, and lower levels of proinflammatory cytokines in the spinal cord at 7 days post-SCI. Immunofluorescence study revealed more CD206 + cells and less iNOS + cells in the injured spinal cord of riluzole-treated SCI rats, as compared to vehicle control. Using real-time PCR, we found that riluzole upregulated the mRNA levels of M2 markers, but downregulated that of M1 markers, as compared to the vehicle treatment. Current findings suggest that systemic administration of riluzole after acute SCI facilitated motor function recovery and inhibited inflammatory responses, which may be associated with polarization of M2 microglia/macrophages., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

The lifetime prevalence of addiction to cannabis (marijuana or cannabinoids) is among the highest of that of all illicit drugs in developed countries, and no effective treatment for cannabis addiction is currently available. Previous studies in our research group suggested that astrocyte-mediated long-term depression (LTD) in ventral tegmental area (VTA) dopamine neurons plays a pivotal role in the development of cannabis addiction. The present study was designed to investigate the effects and potential mechanisms of riluzole, a drug used for the treatment of motor neuron diseases, on cannabinoid-facilitated LTD induction. We demonstrated that riluzole significantly inhibited HU210-induced glutamate release through increased expression of glial glutamate transporter-1 (GLT-1) in cultured astrocytes, and an infusion of riluzole into the bilateral VTA in rats prevented the potent cannabinoid HU210-facilitated LTD induction in 2-month-old animals, which was blocked by bath application of dihydrokainate (DHK), a selective GLT-1 inhibitor. Our results show the effects and potential mechanism of action of riluzole in cannabinoid-facilitated LTD induction, which may provide insights into new therapeutic approaches for cannabis addiction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. So far, no cure exists, prompting studies in disease mechanisms to facilitate development of new treatment strategies. In this study, we employed the wobbler mouse model of ALS focusing on a symptomatic group of animals. We studied the neurophysiological changes conferred by riluzole or diazepam application, two drugs employed in ALS. Riluzole is an antiglutamatergic agent and the only drug to offer some effect on the life expectancy of ALS patients. To target the inhibitory system, we utilized diazepam as a GABAergic modulator. Acute brain slices were prepared from the wobbler mouse model and analyzed using extracellular field recordings in the hippocampus. During Schaffer collateral stimulation, riluzole caused a marked reduction in the paired-pulse ratio (p<0.0001). Importantly, this reduction was more pronounced in wobbler slices (e.g. 184.2±8.9% at 20ms interval without riluzole, and 124.3±9.8% in the presence of riluzole) compared to control slices (at 20ms: from 198.7±5.8% to 160.5±6.7%). Diazepam caused less pronounced effects at wobbler slices and reduced the paired-pulse ratio more in control animals compared to wobbler individuals (p<0.0001). Comparable results were obtained during trains of stimulations (10 pulses at 20Hz). Importantly, paired-pulse ratios as well as synaptic facilitation were overall similar in control and wobbler slices, without the drugs present, indicating that the differences were only revealed pharmacologically. In summary, the present data support excitatory-inhibitory imbalances in the brain of the wobbler mouse and further consolidate this mouse as an animal model of ALS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuropathic pain is becoming an intractable health threat, with its profound effect on quality of life, thus posing a major challenge to clinical therapy. Despite the reported efficacy of riluzole in some pain models, the underlying mechanism remains largely unknown. The present study aimed to assess the effects of riluzole in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Subsequent to model establishment, paw withdrawal latencies (PWLs) and the paw withdrawal mecha threshold (PWT) rapidly decreased, coupled with inhibited microglial activation and upregulated P2X7R expression in the spinal cord dorsal horn (SCDH). Following intraperitoneal administration of riluzole (4mg/kg) once daily for 5 consecutive days as from day 3 after surgery, the mechanical allodynia and thermal hyperalgesia in the hind limbs were significantly attenuated. In addition, riluzole downregulated P2X7R expression and inhibited microglial activation in SCDH. Our results indicated that riluzole is effective in alleviating neuropathic pain and inhibiting microglial activation, presumably via the downregulated P2X7R expression in SCDH., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The roles of persistent Na(+) currents (INaP) in intrinsic membrane properties were examined in rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. In a voltage-clamp mode, riluzole inhibited the slow voltage ramp-induced INaP but had little effect on the peak amplitude of transient Na(+) currents in SG neurons. In a current-clamp mode, most SG neurons exhibited spontaneous action potentials and tonic firing pattern. Riluzole reduced both spontaneous and elicited action potentials in a concentration-dependent manner. The present results suggest that the riluzole-sensitive INaP plays an important role in the excitability of SG neurons and are thus, likely to contribute to the modulation of nociceptive transmission from the orofacial tissues., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5-L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Riluzole is the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS) but its precise mode of action is not properly understood. Damage to axonal transport of neurofilaments is believed to be part of the pathogenic mechanism in ALS and this has been linked to defective glutamate handling and increased phosphorylation of neurofilament side-arm domains. Here, we show that riluzole protects against glutamate-induced slowing of neurofilament transport. Protection is associated with decreased neurofilament side-arm phosphorylation and inhibition of the activities of two neurofilament kinases, ERK and p38 that are activated in ALS. Thus, the anti-glutamatergic properties of riluzole include protection against glutamate-induced changes to neurofilament phosphorylation and transport.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative fatal disease. Drugs used in this disease need to cross the blood-brain barrier (BBB). Only riluzole is approved for ALS treatment. We have investigated riluzole as a breast cancer resistance protein (BCRP) substrate by studying its brain transport in CF1 mdr1a (-/-) mice and its intracellular uptake on BeWo cells (human placental choriocarcinoma cell line). We have also investigated the effect of riluzole on BCRP expression level and on its activity using the prazocin as a test probe for brain transport and intracellular uptake. Assays on mdr1a (-/-) mice and BeWo cells showed a higher uptake of riluzole when pretreated with a BCRP inhibitor. After repeated doses of riluzole, BCRP activity was increased in CF1 mdr1a (-/-) mice, riluzole uptake was decrease and both BCRP expression and activity were increased in BeWo cells. In conclusion, we report in this study that riluzole is transported by BCRP at the BBB level and can enhance its function. These results taken with our previous studies on riluzole and P-glycoprotein show that drug-drug interactions between riluzole and efflux transporters substrates may occur at the BBB level and should be taken into account in future clinical trial design in ALS.

The pre-Bötzinger complex (PBC), part of the ventral respiratory group that is responsible for inspiratory rhythm generation, contains at least two types of pacemaker neurons. In vitro studies have shown that bursting properties of one type of pacemaker relies on a riluzole-sensitive persistent sodium current, whereas bursting of a second type is sensitive to flufenamic acid (FFA), a calcium-dependent nonspecific cationic current blocker. In vitro, under control conditions, the PBC generates fictive eupneic activity that depends on both riluzole-sensitive and FFA-sensitive pacemaker neurons. During hypoxia the PBC generates fictive gasping activity and only riluzole-sensitive pacemaker neurons appear to be necessary for this rhythm. We carried out pharmacological experiments to test the role of respiratory pacemaker neurons in vivo by performing plethysmographic recordings on neonate mice. As reported in vitro, eupnea activity in vivo is abolished only if both FFA and riluzole are coadministered intracisternally, but not when either of them is administered independently. On the other hand riluzole, but not FFA, drastically reduced gasping generation and compromised the ability of mice to autoresucitate. Neither substance P nor forskolin was able to reestablish respiratory activity after riluzole and FFA coapplication. Our results confirm in vitro reports and suggest that eupnea generation in neonates requires a complex neuronal network that includes riluzole- and FFA-sensitive elements and that gasping activity depends mostly on a riluzole-sensitive mechanism.

Huntington's disease (HD) is an autosomal dominant, inherited and fatal neurodegenerative disorder for which there is, at present, no effective treatment or cure. Striatal medium spiny neurons (MSN) are the most sensitive in HD. Dysregulation of glutamate/calcium signaling pathway emerges as a possible cause of striatal MSN neurodegeneration in HD. Here we evaluated five clinically relevant glutamate pathway inhibitors using previously developed in vitro HD model. We found that folic acid, gabapentin and lamotrigine did not protect HD neurons from glutamate-induced cell death, but memantine and riluzole were protective. Our results provide further support to potential use of memantine and riluzole for treatment of HD.

Riluzole is a presynaptic inhibitor of glutamate release with neuroprotective properties. In order to evaluate the effects of riluzole on motor activity in post-traumatic peripheral neuropathy (PTPN), the sciatic nerve of Wistar male rats was exposed monolaterally and subjected to crushing for one min by a surgical forceps. Animals received an intraperitoneal treatment with riluzole (2, 4 or 8 mg/kg per day), diclofenac (5, 10 or 20 mg/kg) or with vehicle for 3 days. Motor activity and coordination was evaluated in a circular open field and in the rotorod test. The treatment with riluzole stimulated ambulation in PTPN rats and improved their motor performance and coordination. The effect of treatment with riluzole on locomotor activity was greater than that of treatment with diclofenac and was dose-dependent. Furthermore, in contrast to vehicle- and diclofenac-treated rats, animals treated with riluzole showed a long-lasting improvement of locomotor activity as it was assessed 7 days after the end of treatment. These findings suggest that riluzole may improve motor performance in PTPN, and this does not depend on its antinociceptive activity. Its neuroprotective properties are possibly involved in this effect.

The protective effects of 2-amino-6-trifluoromethoxy benzothiazole (riluzole), a Na(+) channel blocker with antiglutamatergic activity were investigated in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in mice. The mice were injected intraperitoneally (i.p.) with four administrations of MPTP (10 mg/kg) at 1 h intervals and then the brains were analyzed at 3 and 7 days after the treatments. Dopamine, DOPAC and HVA levels were significantly decreased in the striatum 3 days after MPTP treatments. Riluzole dose-dependently antagonized the MPTP-induced decrease in dopamine, DOPAC and HVA levels in the striatum. MPTP treatment also caused a severe decrease in the amount of nigral tyrosine hydroxylase protein (TH) and microtuble-associated protein 2 (MAP 2) and produced a marked increase in the striatal glial fibrillary acidic protein (GFAP). Our immunohistochemical study with TH and MAP 2 staining showed that riluzole can protect against MPTP-induced neuronal damage in the substantia nigra. Furthermore, riluzole markedly increased the striatal GFAP-positive astrocytes 3 days after MPTP treatments. These results suggest that riluzole is effective against MPTP-induced neurodegeneration of the nigrostriatal dopaminergic neuronal pathway. Our findings also may provide a rationale for the identification of astrocytes as a prominent target for the development of new therapies of Parkinson's disease.

Riluzole is an antiexcitotoxic agent used for the treatment of amyotrophic lateral sclerosis, and reported to have neuroprotective effects in animal models of Parkinson's disease, Huntington's disease and brain ischemia. We investigated the effects of riluzole on synthesis of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in cultured mouse astrocytes. The protein and mRNA levels were measured by enzyme-linked immunosorbent assay and semiquantitative reverse transcription-polymerase chain reaction, respectively. Treatment with riluzole at 100 microg/ml (426 microM) for 24 h increased the contents of NGF, BDNF, and GDNF in the culture medium 109-fold, 2.0-fold and 3.1-fold over the control, respectively. The drug-induced relative mRNA levels of NGF, BDNF, and GDNF were 7.3-fold at 2 h, 2.1-fold at 4 h, and 1.9-fold at 2 h, respectively. These results indicate that riluzole stimulates synthesis of NGF, BDNF and GDNF in cultured astrocytes. Riluzole might exert neuroprotective effects, at least in part, via stimulation of neurotrophic factors.

Growth associated protein-43 (GAP-43) gene induction may be involved in reactive events that follow cerebral ischemic damage. Antagonists of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors are thought to ameliorate functional outcome after ischemic injury. To assess whether glutamate NMDA receptor blockade could alter GAP-43 postischemic induction we performed immunocytochemistry in rat brains that had been subjected to middle cerebral artery occlusion. Cortical cells did not constitutively express GAP-43, yet focal ischemia induced its expression, with an intense signal generated in cells over the lesioned area at 6 h, increasing at 24 h postischemia. This signal was effectively decreased by pretreatment with the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (0.1 mg/kg s.c.), but not by the glutamate release blocker riluzole (8 mg/kg i.v.), suggesting that overactivation of NMDA receptor during ischemia is linked to GAP-43 expression.

The effect of riluzole administration, an antiglutamatergic compound, on the electrophysiological activity of the pallidal complex of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys is compared with those induced by two dosages of levodopa (L-DOPA), the first affording the best clinical alleviation, the second sufficient to induce dyskinesias. Both dosages of L-DOPA reduced sharply the firing frequency of globus pallidus pars internalis (GPi) neurons (respectively, 43.8+/-23.0 and 27.4+/-20.2 vs. 111. 2+/-31.4 Hz), decreased the percentage of bursting cells (respectively, 60.7 and 50.0 vs. 80.3%) and augmented the number of regular cells (respectively, 6.5 and 33.0 vs. 4.8%). Riluzole restored the firing frequency (75.0+/-26.9 Hz) and the firing pattern of the GPi (39.7% bursting, 9.5% regular and 50.8% irregular). These results suggest that the emergence of dyskinesia may well be due to a modification of the neuronal messages transmitted from the GPi to the motor nuclei of the thalamus. Riluzole would represent an interesting alternative to dopamine therapy in Parkinson's disease since it regularizes firing but does not cause dyskinesia.

Previous studies have shown that antagonists at glutamatergic N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) receptors can disrupt the development or expression, respectively, of conditioned place preference (CPP) induced by drugs of abuse. The present study examined the effects of inhibition of presynaptic glutamate release by riluzole on the development of morphine- and amphetamine-induced CPP. Morphine (10 mg/kg), D,L-amphetamine (4 mg/kg) and riluzole (4 mg/kg) itself each produced a significant CPP; however, when riluzole was co-administered with morphine or amphetamine during the conditioning sessions, no CPP developed. It is concluded that non-specific disruption of glutamatergic neurotransmission prevents the development of morphine- and amphetamine-induced CPP.