Oleamide (Cis-9,10-octadecenoamide) is a centrally acting fatty acid amide that belongs to the family of endogenous lipid signaling molecules that includes endocannabinoids, anandamide, N-palmitoylethanolamine, and N-oleoylethanolamine (Fowler, 2004). Oleamide was first found to exist in the cerebrospinal fluid of sleep-deprived animals and act as an endogenous sleep-inducing substance (Cravatt et al., 1995). Besides inducing sleep, systemic administration of exogenous oleamide has been shown to produce a variety of central nervous system (CNS) effects (Boger et al., 2000b; Leggett et al., 2004), including elicitation of hypothermia (Fedorova et al., 2001), analgesia, memory (Murillo-Rodriguez et al., 2001; Akanmu et al., 2007), food intake (Martinez-Gonzalez et al., 2004), hypo-locomotion (Huitron-Resendiz et al., 2001), and reduction of pentylenetetrazole-induced epileptic behavior (Wu et al., 2003; Solomonia et al., 2008). Furthermore, it was recently reported that oleamide reduces amyloid-β (Aβ) accumulation via enhanced microglial phagocytosis and suppresses inflammation after amyloid Aβ deposition (Ano et al., 2015).

Although a wide range of neuropharmacological actions of oleamide have been suggested in several neurotransmitter systems, its effects on brain damage are less well studied and its mechanisms of action remain elusive. The endocannabinoid system is known to play a role in the cell death/survival decision and to improve glutamate homeostasis, thus reducing excitotoxicity (Zoppi et al., 2011). As oleamide is structurally similar to an endogenous fatty acid amide, anandamide, it has been speculated that oleamide possesses full agonist activity on the cannabinoid (CB₁) receptor (Boger et al., 2000a; Leggett et al., 2004). On the other hand, other reports have indicated that oleamide has negligible or no effects on the CB₁ receptor (Mechoulam et al., 1997; Lichtman et al., 2002). Other neuronal receptor systems have also been reported to be associated with the actions of oleamide. Oleamide has shown to inhibit gap junction (connexin)-mediated cell–cell communication (Guan et al., 1997; Boger et al., 1998), and to modulate ionotropic γ-amino butyric acid (GABA_A) receptors (Verdon et al., 2000), and serotonergic 5-HT₁, 5-HT_2A/2C, and 5-HT₇ receptors (Huidobro-Toro and Harris, 1996; Mueller and Driscoll, 2009). However, little is known about the neuroprotective effect of exogenous oleamide against neuronal death or its underlying intracellular mechanisms.

Kainic acid (KA) has been used to produce an epileptic animal model that mimics human temporal lobe epilepsy (Nadler, 1981) and induce sustained neuronal depolarization and hyperexcitability (Frerking et al., 1998), leading to excitotoxicity in various brain regions including striatum, hippocampus, cerebral cortex, amygdala, and nucleus accumbens, etc. (Zaczek et al., 1980; McGeer and Zhu, 1990; Ferrer et al., 1995; Araujo et al., 2008). Excitotoxicity is a major mechanism of neuronal death in acute brain injury such as stroke, epilepsy, and traumatic brain injury, and is also related to chronic neuronal degenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and others (Lau and Tymianski, 2010). As glutamate is the major excitatory neurotransmitter in the CNS, overstimulation of its receptors increases intracellular Ca²⁺ levels by directly opening post-synaptic ion channels and secondarily affecting Ca²⁺ homeostatic mechanisms (Chen et al., 2000). An increase in the cytoplasmic Ca²⁺ level activates a number of Ca²⁺-dependent proteases, particularly calpains, leading to various types of cell death (Araujo et al., 2010) that include necrotic cell death through various different pathways, such as those involving membrane breakdown, cytoskeletal alterations, and nitric-oxide-derived free radicals (Norenberg and Rao, 2007; Mehta et al., 2013), and also triggers apoptosis (Mattson, 2007; Quintanar et al., 2012) or autophagic neuronal death (Kim et al., 2009). Therefore, interest in calpain inhibitors has been growing in an effort to overcome the calpain activity related to cell death that plays a main role in such a wide variety of CNS disorders. The high concentration and activity of calpain that accompanies neuronal degeneration has been seen in the brains of epileptic animal models (Bi et al., 1997; Feng et al., 2011), and KA-induced excitotoxic injury also appear to be prevented by calpain inhibitors (Fitzpatrick et al., 1992).

The present study investigates the antiepileptic and neuroprotective effects of oleamide through calpain inhibition as a potential intracellular mechanism. The ability of oleamide to inhibit calpain activity was demonstrated in both brain tissues of KA-induced in vivo epileptic rat models and in vitro neuronal systems.

The present study investigates the in vivo antiepileptic and neuroprotective effects of exogenous oleamide against KA-induced excitotoxic brain damage, and asks whether calpain inhibition is an intracellular mechanism of those effects. We demonstrated the preventive effects of oleamide against epileptic behavior in KA-induced epileptic rat model and against excitotoxicity-induced calpain activation that leads to neuronal death. The ability of oleamide to inhibit calpain activity was examined in both brain tissue of a KA-induced epileptic rat model and cultured neurons.

Oleamide is an endogenous fatty acid amide and shares a structure and some characteristics with the endocannabinoid anandamide. Both oleamide and anandamide are degraded by fatty acid amide hydrolase (Boger et al., 2000a). While many biological effects of oleamide are documented, its molecular and signaling mechanisms are less well defined than those of cannabinoids. The endocannabinoid system has been identified as a potential target for the treatment of several disorders of the CNS, including epilepsy and excitotoxicity (Marsicano et al., 2003; Monory et al., 2006). An increasing number of studies have implicated the endogenous fatty acid amide system as a new target for neuronal damage, mainly studies on anandamide and fatty acid amide hydrolase inhibitors (Karanian et al., 2007; Naidoo et al., 2012). Several studies have shown that endocannabinoid deficiency may contribute to the pathophysiology of chronic pain including migraine and inflammatory pain (Lastres-Becker et al., 2002; Clapper et al., 2010; Nozaki et al., 2015). The novel fatty acid amide hydrolase inhibitor, URB-597, has been demonstrated to produce anticonvulsant effect on seizures induced by pentylenetetrazole in rats (Vilela et al., 2013), and to block neuronal hyperactivity in neurons (Nozaki et al., 2015), which can be considered a similar mechanism to that seen in epilepsy. Interestingly, the administration of an endocannabinoid uptake inhibitor (AM404) that kept the fatty acid amide levels high to the rats injected intrastriatally with 3-nitropropionic acid, a toxin that selectively damages striatal GABAergic neurons, was shown to attenuate hyperactive motor disturbances (Lastres-Becker et al., 2002). These reports suggest that fatty acid amides can play important roles as signaling molecules that are involved in mechanisms for epilepsy and motor hyperactivity. Another fatty acid amide hydrolase inhibitor (AM374) that was reported to cause a prolonged elevation of the anandamide level in the brain reduced KA-induced seizures by promoting CB₁ receptor signaling (Karanian et al., 2007; Shubina et al., 2015). Thus, fatty acid amide hydrolase inhibitors have been suggested to be relevant to the excitotoxic protection by enhancement of the endocannabinoid system, which is elicited via inhibition of endocannabinoid degradation.

Although oleamide is an endogenous fatty acid amide, which is degraded by fatty acid amide hydrolase, only a few of studies have reported on the in vivo antiepileptic and neuroprotective effects by exogenously administered oleamide. We first demonstrated that oral administration of oleamide significantly reduced seizure behavior in KA-induced epileptic rats. The KA-induced chronic epileptic animal model has been known to mimic human temporal lobe epilepsy and status epilepticus (Nadler, 1981). Many reports have shown that changes in the hippocampus and/or cortex are mainly involved in epileptic seizures. The striatum, however, is also well reported as an important brain region of the neuronal damage with relation to the occurrence of convulsions, including KA-induced seizures in rats (Pisa et al., 1980). In rats induced epilepsy by injection of KA into the striatum, anti-seizure effect of oleamide was more potent than that of carbamazepine, a clinically long-used anticonvulsant agent (Figure 1). Previously, the anticonvulsant effect of oleamide was only shown to produce against the behavioral seizures induced by pentylenetetrazole, the most commonly used acute seizure model (Wu et al., 2003; Solomonia et al., 2008). According to the Wu et al.’s (2003) report, intraperitoneal administration of oleamide (43.8–700 mg/kg) to mice significantly attenuated the latency of seizure onset induced by pentylenetetrazole (85 mg/kg, i.p.) that was administered 30 min after the injection of oleamide, but produced no effect on seizures induced by other convulsive agents, such as picrotoxin, semicarbazide, strychnine, and caffeine. This report does not fully explain the selective action of oleamide on pentylenetetrazole-induced seizures, and the in vivo dose range of oleamide used in the report is exceptionally higher than that reported by other researchers. In another short report, oleamide in a dose of 10 mg/kg injected (i.p.) to rats was shown to produce anti-seizure effect specifically on the degree (severity) of convulsions, but not decrease the duration and latency of convulsions (Solomonia et al., 2008).

In addition, we found that oleamide prevents KA-induced neuronal death through histological analyses of cellular integrity in striatal sections of KA-induced epileptic rats (Figures 2, 3). The striatum has been demonstrated to express a large number of binding sites for all classes of glutamate receptors (Albin et al., 1992; Wullner et al., 1994; Miyamoto et al., 2008), and the intrastriatal injection of KA has been to lead to substantial neuronal loss in striatal tissues (Wang et al., 2006). Apoptotic and necrotic death of neurons is involved in KA-induced excitotoxicity in vivo (Wang et al., 2005; Vincent and Mulle, 2009). The KA-induced striatal damage observed in our demonstrated experimental condition can be considered from direct toxic effect of KA, presumably through excessive stimulation of striatal glutamate receptors, since the neuronal damage was observed in only right side of striatum where KA was directly injected, but not in saline-injected left side of striatum. Excessive stimulation of glutamate receptors and neuronal damage due to KA is thought to be a result of a large influx of Ca²⁺ into neurons and by a dysfunction of downstream signaling systems (Koh et al., 1990; Lipton and Rosenberg, 1994) including calpain activation.

It has been reported that neuronal damage can spread from the KA-injected striatum into contiguous structures (Zaczek et al., 1980), and can induce locomotor changes and epileptogenesis (McGeer and Zhu, 1990). Although KA-induced seizures are followed by extensive neurodegenerative changes, the seizure-induced brain damage is still debated (not conclusively demonstrated) both in humans and in animal models of epilepsy (Rossini et al., 2017).

Alterations in Ca²⁺ homeostasis lead to persistent, pathologic overactivation of calpain in a number of neurodegenerative diseases (Vosler et al., 2008). Initially, calpain activation was thought to cause only necrotic cell death, while activation of caspase led to programmed cell death. There is a shared mechanism of calpain activation in neurodegenerative diseases, such as epilepsy, Alzheimer’s disease, and Parkinson’s disease (Lau and Tymianski, 2010; Lopatniuk and Witkowski, 2011). In particular, calpain-specific cleavage of Cdk5-p35 to p25 has been implicated in the neurological damage seen in many neurological disorders (Kanungo et al., 2009; Cheung and Ip, 2012). Calpains have long been implicated in neuronal cell death induced by triggers of neuronal injury including excitotoxicity. Putkonen et al.’s (2011) reported that KA produced a dose-dependent increase in intracellular Ca²⁺concentration and raised calpain activity, followed by induction of phosphorylation of Cdk5 and cleavage of Cdk-p35, which are believed to be involved in KA-mediated degeneration of glutamatergic synapses in the rat hippocampus (Putkonen et al., 2011). Also in the striatum, it has been shown that intrastriatal injection of KA increases calpain mRNA (Campbell et al., 2004). In addition, calpain activation is involved in manganese-induced neuronal death in rat striatum (Quintanar et al., 2012).

Since many calpain inhibitors have been shown to produce antiepileptic effects (Lubisch et al., 2000), we postulated that oleamide can also affect calpain activity, and investigated cleavage of the representative calpain substrate protein, Cdk-p35 in KA-induced epileptic rat striatal tissues. We found that the KA-induced enhancement of Cdk5-p35 cleavage to p25 was significantly blocked by oral administration of oleamide (Figure 4A), implying that oleamide could produce antiepileptic and neuroprotective effects by the mechanism related to calpain inhibition.

The KA-induced significant neuronal loss can lead to the axonal degeneration that is preceded by disruption of Ca²⁺ homeostasis, causing calpain activation and the proteolytic degradation of axonal proteins (Saatman et al., 1996). CRMP-2 is known to be involved in neuronal differentiation and control of neuronal polarity and axonal outgrowth (Yoshimura et al., 2006). In addition, more recent studies have reported that CRMP-2 can bind to the voltage-gated calcium channel Cav2.2, and this interaction may play a crucial role in neurotransmitter release from the presynaptic terminals of hippocampal neurons (Wang et al., 2010). In our previous study, CRMP-2 protein was shown to be altered into a cleaved form with a size of 58 kDa in the brain under ischemic conditions (Chung et al., 2005), which was later shown to be induced by calpain activation (Hou et al., 2009). Since then, it has been discovered that CRMPs are cleaved in severe neurodegenerative conditions and their expression levels are changed in several neuronal diseases. CRMP-2 was reduced in patients with temporal lobe epilepsy (Czech et al., 2004). A relatively new antiepileptic drug, lacosamide, was reported to modulate CRMP-2 and inactivate voltage-gated sodium channels, which were identified as its antiepileptic mechanisms (Saussele, 2008; Patyar and Medhi, 2010). In the present study, calpain-induced CRMP-2 cleavage was observed in KA-induced epileptic rat striatal extracts, and, similar to Cdk5-p35, oleamide significantly blocked CRMP-2 cleavage (Figure 4B). Additional evidence implying that oleamide can improve KA-induced synaptic dysfunction was provided by measuring the changes in the level of synapsin-II, one of the synaptic marker proteins. KA dramatically reduced the level of synapsin-II, and oleamide dose-dependently protected the synapsin-II levels (Figure 4C). These data indicate that oleamide can block the calpain-mediated cleavage of substrate proteins that play important roles in the regulation of neuronal survival/death and neuronal activity.

We further confirmed the calpain inhibitory effects of oleamide by examining changes in endogenous calpastatin levels. An endogenous calpain inhibitory protein, calpastatin, has been proposed to play pro-survival roles in adult neurons under degenerative conditions, including in models of ischemia–excitotoxicity (Yang et al., 2013). Recently, exogenous transgenic expression of calpastatin was used to provide definitive evidence for calpain’s involvement in sciatic and optic nerve degeneration after sectioning in vivo (Ma et al., 2013). Data (Figure 4D) showing the ability of oleamide to enhance the level of endogenous calpastatin, which is reduced in KA-induced epileptic rat brain, suggest that its antiepileptic and possible neuroprotective effects are due to calpain inhibition.

So far, oleamide was assumed to exert its various biological actions through as-yet undefined membrane receptors. The calpain inhibitory effect of oleamide has not been reported previously. We hypothesized that oleamide might play a role as a calpain inhibitor. To investigate this, in vitro experiments were performed. In calpain-transfected cells, oleamide was observed to decrease the μ-calpain activity more effectively than the known calpain inhibitors E64d and calpeptin (Figure 5). In addition, the CRMP-2 and Cdk5 degradation observed in rat brain tissue lysates directly incubated with μ-calpain were inhibited by exogenous treatment with oleamide (Figure 6). These results provide evidence for the possibility of direct calpain inhibition by oleamide. It can be speculated that oleamide may enter into the intracellular space through an endogenous fatty acid amide membrane transporter that has been shown to transport anandamide (Mechoulam and Deutsch, 2005), and then inhibit calpain directly and/or indirectly, however, evidence for this theory is still lacking.

Taken together, our findings reveal that exogenous oleamide attenuates not only KA-induced behavioral seizures but also calpain-mediated neuronal cell death in the brain, suggesting that oleamide is a novel promising drug candidate for various neuronal diseases including epilepsy. In addition, the antiepileptic and neuroprotective effects of oleamide could be mediated through a novel mechanism by calpain inhibition, although its direct calpain inhibitory effect needs to be further pursued.

HYN performed experiments, analyzed the data, and wrote the first draft of manuscript. EJN, EL, and YK analyzed data and involved in the manuscript preparation. H-JK designed and supervised the research, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.