Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
CRITICAL REVIEW AND INVITED COMMENTARY
Ghrelin: An emerging new anticonvulsant neuropeptide Jeanelle Portelli, Yvette Michotte, and Ilse Smolders Center for Neurosciences, Department of Pharmaceutical Chemistry & Drug Analysis, Vrije Universiteit Brussel, Brussels, Belgium
SUMMARY Neuropeptides appear to be of importance when the central nervous system (CNS) is challenged, such as during high-frequency firing and pathologic conditions. Potential advantages of treatments that target neuropeptide systems in comparison to classical neurotransmitter systems and ion channels revolve around the subject of efficacy as well as the reduced likelihood of side effects, thus making them attractive candidates for the development of new clinical applications for various disorders. The number of
The search for new drugs is ongoing, and the process of drug discovery has never been easy. Only a small percentage of promising candidates manage to arrive at the clinical stages, with a percentage of these being withdrawn before reaching the market. Researchers are continuously on the search for new, innovative ways to treat certain diseases/ conditions that up to now do not provide adequate response to the available drugs. In recent years considerable interest has been shown toward neuropeptides as a way to suppress seizures.
Neuropeptides as Potential Targets for Peptide-Based Epilepsy Therapy Neuropeptides are usually about 3–100 amino acid residues long. They are smaller and have a less complex threedimensional structure than regular proteins. The great majority of neuropeptide receptors are of the seven transmembrane receptor family, better known as the G protein– coupled receptor (GPCR) family. Both GPCRs and their ligands are widely distributed in both the central nervous system (CNS) and peripherally. The role of neuropeptides in the CNS has been studied for decades, and neuropeptides commonly occur with (and also complement to) the classic neurotransmitters. A number of differences are presAccepted January 11, 2012; Early View publication March 14, 2012. Address correspondence to Prof. Dr. Ilse Smolders, Laarbeeklaan 103, 1090 Brussels, Belgium. E-mail:
[email protected] Wiley Periodicals, Inc. ª 2012 International League Against Epilepsy
neuropeptides linked to epilepsy is on the rise, reflecting the increased interest of researchers in this domain. Ghrelin has only very recently been introduced into the field of epilepsy, and has already led to contradictory clinical publications. There is a great paucity with regard to what mechanism of action is utilized by ghrelin to inhibit seizures. In this review we disclose how we can better understand the mechanism ghrelin uses to prevent seizures, which indirectly could give an insight to researchers who are studying ghrelin in other fields of research. KEY WORDS: Epilepsy, Hippocampus, Peptide, Memory.
ent between peptides and other messenger molecules, which are amply described in the review by Hokfelt et al. (2003). A number of neuropeptides have been implicated in the pathogenesis of epilepsy, such as neuropeptide Y (NPY), somatostatin, and galanin. Potential advantages of treatments that target neuropeptide systems in comparison to classical neurotransmitter systems and ion channels revolve around the subject of efficacy as well as safety. Because the occurrence of side effects is dependent on both selectivity and potency of a ligand for its receptor, it is expected that targeting neuropeptide receptors might result in lesspronounced side effects when compared to classical neurotransmitters. Indeed, neuropeptides have more discrete neuroanatomic localization when compared to classic neurotransmitters. They have as well an even higher binding affinity for their receptors than neurotransmitters (nM vs. lM, respectively), being consequently more potent. In addition, another possible advantage could be that because neuropeptides are normally released from neurons in the presence of high frequency firing or pathologic conditions, the clinical effects of neuropeptide receptor antagonists will become evident only under epileptic conditions where high frequency firing is involved, consequently reducing the risk of side effects. Neuropeptides possess additional differences relative to classic transmitters: they contain more chemical information than classic transmitters and have more recognition sites for receptor binding as well as the availability of transgenic models to study neuropeptides, since neuropeptides are encoded in the genome (Hokfelt et al., 2003). In recent years, the neuropeptide ghrelin has emerged as a promising candidate in the field of epilepsy. Kojima (2008), one of the trio that discovered ghrelin, gives an interesting
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586 J. Portelli et al. rendition of how ghrelin was discovered. Ghrelin’s discovery was a case of reverse pharmacology, since the mission was to establish the endogenous ligand of the growth hormone secretagogue receptor (GHSR). It proved to be a tantalizing task that took several months, relocation from the brain to the stomach, and weeks of trying to determine the modified structure that was essential for ghrelin’s activity. Today, following ghrelin’s discovery in 1999, a simple search on PubMed results in about 5,000 publications involving this neuropeptide; therefore, it is safe to say that ghrelin has been an exciting neuropeptide and hormone to explore over the last decade.
Ghrelin and Its Receptor(s) Ghrelin is mainly produced by the X/A-like cells of the oxyntic stomach mucosa and centrally by the neurons in the hypothalamus. The ghrelin gene is located in chromosome 3p25-26, and the main messenger RNA (mRNA) codes for the 117 amino acid preproghrelin, which is consequently cleaved enzymatically into proghrelin and obestatin (Lim et al., 2011). There are two forms of circulating ghrelin—acylated ghrelin and des-acyl ghrelin. Ghrelin requires modification on the serine-3 by O-acylation with octanoate to execute its endocrine actions. In other words, this acylation is a posttranscriptional modification that is required for its binding to the GHSR type 1a (GHSR1a) (Kojima et al., 1999). For several years researchers tried to identify the mechanism by which ghrelin is acylated in vivo. In 2008, two separate groups identified the enzyme responsible for attaching the octanoate to serine-3 of ghrelin as ghrelin-Oacyltransferase (GOAT) (Gutierrez et al., 2008; Yang et al., 2008). Ghrelin is a 28–amino acid motilin-related and orexigenic peptide. Across the mammalian species, the amino acid sequence of ghrelin is well conserved (Table 1). In fact, the 10 amino acids in the NH2 termini of mammalian ghrelins are identical. It is interesting to note that the serine-3 is present in mammals, fish, and birds, whereas in the bullfrog the octanoylation of serine-3 is replaced by an octanoylation of threonine (Kojima & Kangawa, 2005). This denotes the significance of the O-octanoylation of ghrelin, since it has been ably conserved in mammals and nonmammals alike throughout the evolutionary process. Such conservation is also true for GHSR1a, since it is detectable as far back as zebrafish (Olsson et al., 2008). The ghrelin receptor gene encodes two types of GHSR mRNA, known as 1a and 1b, resulting in two isoforms of GHSR: GHSR1a and GHSR1b (Camina, 2006). Importance is given ascribed to GHSR1a, since GHSR1b is unable to bind or be activated by ghrelin. GHSR1a is widely expressed both centrally and peripherally, including in seizure-prone regions such as the hippocampus. GHSR1a is a GPCR that belongs to a small family of receptors for peptide hormones and neuropeptides. By way of the Ga11/Gq11-protein, Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
GHSR1a leads to depolarization by activating the phospholipase C (PLC) pathway, which results in intracellular Ca2+ increases followed by K+ channel inhibition (Ferrini et al., 2009). For a more detailed account of the cell biology of the ghrelin receptor, refer to the review by Camina (2006). Ghrelin is a pleiotropic peptide that has gained a lot of attention as a brain-gut hormone. Ghrelin is best known for its role in feeding behavior and metabolism (Ghigo et al., 2005). Ghrelin also affects other physiologic processes in the body, such as pituitary hormone secretion, the cardiovascular system, the autonomic nervous system, the immune system, the musculoskeletal system, sleep regulation, and very recently epilepsy (Angelidis et al., 2010). GHSR expression and ghrelin-binding sites are present in seizure prone regions of the rodent brain, such as hippocampus and cerebral cortex (Cowley et al., 2003; Diano et al., 2006). Humans can tolerate intravenous injections of ghrelin rather well (Akamizu et al., 2004). In human plasma, acylated ghrelin was found to disappear more quickly than total ghrelin, with elimination half-lives of 9–13 and 27–31 min, respectively (Akamizu et al., 2004). A number of synthetic ghrelin agonists are currently being investigated, with compounds such as ARD-07 (EP01572) showing a promising pharmacokinetic profile with regard to its significantly longer elimination half-life and good tolerability in human subjects (Piccoli et al., 2007; MacLean et al., 2009).
Ghrelin and Epilepsy: Human Studies A number of human studies have attempted to comprehend the link between ghrelin and epilepsy, resulting in a notable disparity to which direction ghrelin levels are
587 Ghrelin and Epilepsy altered in patients with epilepsy. As presented in Table 2, clinical studies have shown increased or decreased levels of ghrelin. This led to the perception that human studies in relation to ghrelin are controversial; however, a closer look indicates that this may not be necessarily so. Too many variables are present among these studies, which can ultimately lead to confusion. For instance variations of ghrelin levels with age are known to take place, thus one has to distinguish between results obtained from the prepubertal and pubertal groups (Whatmore et al., 2003). Indeed, the Gungor et al. (2007) detected that although serum ghrelin levels increased significantly following 6 months of treatment in prepubertal children, no changes were detected in the pubertal group. Ideally, ghrelin levels in patients who are undergoing antiepileptic drug (AED) therapy should be evaluated against the same cohort before drug treatment and not against a healthy control group. This will allow correct evaluation of AED-induced ghrelin changes in patients with epilepsy. Caution must be applied when interpreting results from different antiepileptic drugs (AEDs) due to an indirect effect on ghrelin levels. It has been suggested that valproic acid (VPA) treatment could affect ghrelin stimulation in a manner different from that of other AEDs, and prolonged
treatment could lead to higher ghrelin levels (Berilgen et al., 2006; Prodam et al., 2010). Moreover, decreases in serum ghrelin levels were observed in VPA-treated children but not in oxcarbazepine-treated children (Cansu et al., 2011a,b). The effect of VPA on ghrelin levels may be due merely to its ability to induce weight gain in treated subjects. To accurately determine whether ghrelin levels are altered in patients with epilepsy, preferably following an epileptic episode, ghrelin levels in control healthy subjects should be compared to those in patients with epilepsy who are undergoing no AED treatment. Because of ethical constraints, not all current ghrelin human studies encompass an epileptic nontreated group. In an interesting study by Aydin et al. (2011), venous blood samples were taken at different time points for 48 h in patients who were undergoing no AED treatment for the duration of the study. This led to the finding that both the acylated and des-acyl forms of ghrelin were significantly decreased within 5 min of a seizure when compared to healthy control subjects. This is in line with previous findings from the same group (Aydin et al., 2009; Dag et al., 2010), as well as what was observed in animals (Ataie et al., 2011), whereas these results contrasted with the findings of Greco et al. (2005) since this group observed
Table 2. Overview of ghrelin level alterations in human epilepsy Study
Patient group
AED Treatment
Component analyzed
Ghrelin form analyzed
Ghrelin levels fl in weight gain patients „ in nonweight-gain patients › in all epilepsy patients › partial epilepsy group > › 1 generalized epilepsy group › prepubertal patients „ pubertal patients fl in both ghrelin forms in all epilepsy patients Ghrelin levels recovered following treatment, but still remained lower than control values
Greco et al. (2005)
Postpubertal females
VPA
Blood serum
NA
Berilgen et al. (2006)
Adult males + females
VPA, PHT, CBZ
Blood serum
Gungor et al. (2007) Aydin et al. (2009)
Children 3–15 years Adult males + females
VPA
Blood serum
Total ghrelin (des-acyl + acylated ghrelin) NA
VPA, PHT, CBZ
Blood serum Saliva
Des-acyl form Acylated form
Prodam et al. (2010)
VPA, CBZ
Blood plasma
Total ghrelin
fl in epileptic patients fl in CBZ grp > fl VPA grp
Dag et al. (2010)
Children CBZ grp: 5.6 ± 0.4 years VPA grp: 5.0 ± 0.4 years Adult males + females
VPA, PHT, CBZ
Blood serum Saliva
Total ghrelin
Aydin et al. (2011)
Adult males 20 ± 0 years
Blood serum
Des-acyl form Acylated form
Cansu et al. (2011a) Cansu et al. (2011b)
Children ±10 years Children 8.8 ± 3.6 years
VPA, PHT, CBZ stopped 48 h prior study initiation VPA
Blood serum
Total ghrelin
fl in serum + saliva of epileptic patients fl partial epilepsy group < fl 1 generalized epilepsy group fl in both ghrelin forms 5 min and 1 h of a seizure. Levels increased with time fl in weight gain patients
OXC
Blood serum
Total ghrelin
„
VPA, valproic acid; PHT, phenytoin; CBZ, carbamazepine; OXC, oxcarbazepine; NA, not available; grp, group; 1, primary;›, increased; fl, decreased; „ , unchanged.
Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
588 J. Portelli et al. no alterations in total ghrelin levels. With this knowledge, the general consensus is that ghrelin levels have the tendency to decrease following epileptic episodes.
Ghrelin and Epilepsy: Animal Studies Published rodent studies are simpler to interpret, since the majority state that ghrelin has anticonvulsant properties (Obay et al., 2007; Aslan et al., 2009; Lee et al., 2010). The group of Obay noted that intraperitoneal (i.p.) injections of ghrelin, with doses ranging from 20–80 lg/kg, successfully delayed or prevented the development of pentylenetetrazole (PTZ)–induced epileptic seizures in rats (Obay et al., 2007). Since in hypothalamic slice preparations it was found that ghrelin leads to increased activity of NPY/AgRP (agoutirelated protein) neurons as well as an increased rate of c-aminobutyric acid (GABA) secretion (Cowley et al., 2003), the group of Obay proposed this stimulatory effect of ghrelin as a possible mechanism of action of ghrelin’s anticonvulsant effects (Obay et al., 2007). Obay et al. (2008) also showed that oxidative stress, which is known to increase in epileptic seizures, was found to diminish when rats were pretreated with ghrelin before PTZ administration. The role of the nitric oxide (NO) pathway in epilepsy is rather unclear, having researchers showing either anticonvulsant or proconvulsant effects (Ferraro & Sardo, 2004). This uncertainty is thought to be due to a number of factors, namely the model selected, dosage of the used convulsant, and the applied NO synthase (NOS) inhibitors. A number of studies show the possible association of NO pathways in ghrelin-mediated effects (Gaskin et al., 2003; Xu et al., 2008). In light of this, Aslan et al. (2009) looked into the ghrelin-NO pathway in penicillin-induced epileptiform activity. They concluded that intracerebroventricular (i.c.v.) ghrelin diminishes the frequency of epileptiform activity and requires activation of endothelial-NOS/NO route in the brain. The pilocarpine model is a commonly used chemoconvulsant-induced model for temporal lobe epilepsy (TLE). A recent ex vivo study showed that ghrelin was also found to possess neuroprotective properties on pilocarpine-induced seizures by promoting the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and so reversing the decreased ratio of Bcl-2 to Bax induced by seizures, and inhibiting caspase-3 activation (Xu et al., 2009). Xu et al. could not confirm whether the neuroprotective effects of ghrelin were due to its action on GHSR1a or another unknown receptor, since GHSR1a mRNA and protein levels showed no significant changes 24 h following seizure induction, and ghrelin treatment had no effect on GHSR1a expression. This is not surprising given that some studies have already indicated the possibility that ghrelin’s antiaEpilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
poptotic effects were independent of GHSR1a (Delhanty et al., 2007; Granata et al., 2007). When this review was written there was only one study where there were attempts to block the anticonvulsant effect of ghrelin via an antagonist. Lee et al. (2010) claim to have been successful in blocking the effect of ghrelin on kainic acid (KA)–induced seizure activity using the ghrelin receptor antagonist D-Lys3-GHRP6. They also noted that ghrelin showed antiapoptopic and antiinflammatory effects in KA-induced hippocampal neurodegeneration through GHSR1a activation. A recent study by Biagini et al. (2011) had a different take on ghrelin’s properties in epileptic mechanisms. The authors note that ghrelin was unable to prevent seizures induced by kainic acid or pilocarpine. They also claim that des-acyl ghrelin prevented status epilepticus in the majority of pilocarpine-treated rats as well as significantly delayed the onset of status epilepticus in kainate-treated rats. Although these results are of interest, certainly some drawbacks of this study are the low number of rats tested and the claim that both ghrelin and des-acyl ghrelin improved survival in pilocarpine-treated rats, since there was already a low mortality rate present in the saline group. From this study alone it is premature to allege that ghrelin has a limited anticonvulsant activity in models of limbic seizures. More investigations need to be performed to better elucidate whether ghrelin is protective against seizures, and consequently against which type of epileptic seizures.
Brain Functions of Ghrelin and Their Relation to Epilepsy Hippocampal synaptic plasticity The hippocampus plays a major role in memory formation, and repeated seizures can result in memory impairment. It has long been regarded that synaptic plasticity changes, notably long-term potentiation (LTP), is of great importance in learning and memory processes (Bliss & Collingridge, 1993). In human patients it was shown that limbic seizures altered hippocampal synaptic plasticity, resulting in LTP impairment (Beck et al., 2000). Recently, Ben-Ari et al. (2008) inquired about whether ‘‘seizures beget seizures’’ in TLE by looking at what evidence has been established. They note that indeed seizures do lead to more seizures as a consequence of several factors, including plasticity changes in the hippocampal network. It is thus a challenge to find a drug that could efficiently attenuate seizures as well as prohibit memory impairment in patients with TLE. Area CA1 of the hippocampus is one of the affected regions in human TLE. As shown by Lehmann et al. (2000) in both human TLE and in pilocarpine-treated rats there is an increase in axon collaterals of CA1 pyramidal cells, or, in other words, alterations of neuronal connectivity in area
589 Ghrelin and Epilepsy CA1 of the hippocampus. Of interest, ghrelin was found to promote both LTP generation in hippocampal slices and the formation of spine synapses in the stratum radiatum of the hippocampal CA1 subfield, which together are known to positively correlate with spatial memory and learning (Diano et al., 2006). Following this finding, Diano’s group tested ghrelin knockout (KO) and wild-type (WT) mice for long-term memory recognition via the novel object recognition test. As expected they found that although WT mice spent more time exploring the novel object on the second day of the test, ghrelin KO mice divided their time equally between exploring the familiar and novel object, which pointed toward the disruption of nonspatial memory. Following exogenous ghrelin administration, the ghrelin KO mice behaved like the WT mice. This was not the first time that ghrelin was linked to learning and memory for it was first reported back in 2002 that i.c.v. ghrelin administration in rats may result in a beneficial effect on memory retention (Carlini et al., 2002). Moreover, the group of Chen showed that a single infusion of ghrelin into the hippocampus not only resulted in long-lasting potentiation of excitatory postsynaptic potentials (EPSPs) but also into population spikes in the dentate gyrus of anaesthetized rats (Chen et al., 2011). Population spikes from the hippocampus are considered to be excitatory and unanimous with epileptiform activity (Smolders et al., 2002), which comes as a surprise given that to date the majority of rodent epilepsy models reveal ghrelin’s anticonvulsant potential. This study further pointed out that although it is believed that activation of the N-methylD-aspartate (NMDA) receptor is required for the expression of LTP in the dentate gyrus, ghrelin-induced potentiation was not attenuated by D-2-amino-5-phosphonovaleric acid (D-APV) (a competitive NMDA receptor antagonist) but by a PI3K inhibitor. As mentioned previously in this review, Xu et al. (2009) state that ghrelin prevents hippocampal neuronal apoptosis triggered by pilocarpine via the regulation of the PI3K pathway. This could mean that ghrelin, apart from being neuroprotective, could lead to a decrease in memory impairment resulting from epileptic seizures. All in all, these observations point to the importance of ghrelin in promoting memory formation. Chronic models of epilepsy should be performed to investigate the antiepileptogenic properties of ghrelin together with memory preservation. Modulatory effects on central neurotransmission In the hypothalamic arcuate nucleus, ghrelin promotes the presynaptic release of NPY and consequently of GABA (Cowley et al., 2003). Moreover, in the paraventricular nucleus, ghrelin reduces the inhibitory tone on corticotropin-releasing hormone (CRH) neurons through presynaptic NPY Y1 and Y5 receptor activation, thus stimulating greater CRH release (Ferrini et al., 2009). As mentioned earlier in this review, since NPY and GABA are known to have inhibitory effects on seizures, it has been suggested that ghrelin’s
anticonvulsant effect may be related to this modulation of neurotransmission. This hypothesis needs further proof, since to the best of our knowledge these ghrelin-NPY/ GABA interactions were described only in the hypothalamic circuitry and not in the cortical or thalamic brain regions, which are involved mainly in the generation of PTZ-induced seizures (van Luijtelaar & Sitnikova, 2006). It is well accepted that classical neurotransmitter systems can enhance or decrease the threshold for seizure susceptibility. Various groups have shown that dopamine lowers seizure thresholds via activation of dopamine D1-type receptors. Cifelli & Grace (2011) investigated whether pilocarpine-induced spontaneous seizures in the rat induce alterations in mesolimbic dopamine neuron activity. They found that in the majority of rats there was a significant increase in the number of dopamine neurons firing per electrode track that occurred together with an increase in amphetaminestimulated locomotor activity, thus concluding that TLEassociated psychosis could be due to abnormal hippocampal overdrive of dopamine neuron activity. Brain regions where GHSR1a is coexpressed with D1 receptors include the ventral tegmental area and hippocampus, and ghrelin was shown to have the capacity of amplifying hippocampal D1 receptor–mediated signalling (Jiang et al., 2006) and extracellular concentrations of accumbal dopamine (Jerlhag et al., 2006). This implies that GHSR1a inhibition could provide inhibition of epileptic seizures. Therefore the question arises on whether GHSR1a’s connection with D1 receptors is of importance in epilepsy mechanisms. Neuroprotection and antiinflammation The aim of neuroprotection in status epilepticus is to protect not only from neuronal death but also from neuronal and network dysfunction. Signaling pathways, such as the PI3K and the extracellular signal regulated kinase 1/2 (ERK1/2), are important for neuroprotection (Walker, 2007). Ghrelin has been shown to exert neuroprotective effects both peripherally and centrally (Ferrini et al., 2009), and it has been suggested that ghrelin is capable of stimulating the ERK1/2 and PI3K/Akt pathways (Chung et al., 2008). Therefore it does not come as a complete surprise when ghrelin was found to significantly attenuate pilocarpine-induced neuronal loss in hippocampal CA1 and CA3 regions (Xu et al., 2009). The same group reported that ghrelin upregulated the seizure-induced decreased levels of phospho-PI3K p85 and phosphor-Akt in the hippocampus, and reversed the decreased Bcl-2 level and the increased Bax level at 24 h after hippocampal pilocarpine treatment. Pilocarpineinduced caspase-3 activation was also inhibited by ghrelin (Xu et al., 2009). This was in line with the neuroprotective effects of ghrelin in hippocampal KA-induced seizures. Lee et al. (2010) demonstrated that ghrelin pretreatment significantly reduced hippocampal neuronal cell death, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, as well as caspase-3 expression. Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
590 J. Portelli et al. There is increasing evidence that brain inflammation endorses epileptogenesis (Ravizza et al., 2011). Ghrelin has been shown to act as a potent antiinflammatory mediator in vivo and in vitro, such as in energy balance; endotoxemia and sepsis; pancreatic, hepatic, and kidney disease; brain injury and pain (Baatar et al., 2011; Cheyuo et al., 2011; Stevanovic et al., 2011). The first cells to produce cytokines during seizures are microglia and astrocytes, making them the main sources of proinflammatory molecules in the brain. The inflammatory process subsequently involves neurons and endothelial cells of the blood–brain barrier (BBB) (Ravizza et al., 2011). In epilepsy settings, ghrelin significantly reduced the accumulation of reactive microglia and astrocytes in the hippocampus following KA-induced excitotoxic injury (Lee et al., 2010). In relation to this, Mac-1 (a specific marker for microglial activation) and GFAP (a marker protein for astrogliosis) KA-induced increases in the CA1 and CA3 of the hippocampus were potently suppressed by ghrelin. Lee et al. (2010) also showed that ghrelin inhibited KA-induced increases of tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, cyclooxidase (COX)-2 immunoreactivities as well as matrix metalloproteinase-3 (Mmp3) expression in the hippocampus. These results, together with what is known about the neuroprotective effect of ghrelin in other brain disorders, are promising and call for further investigations in order to fully understand the neuroprotective role of ghrelin in epilepsy and epileptogenesis.
Ghrelin in Comparison with NPY, Somatostatin, and Galanin Table 3 features some basic properties of ghrelin in comparison to NPY, somatostatin, and galanin with regard to structure, receptor types, and receptors involved in attenuating seizures. The amino acid sequence for all four neuropeptides shows a high degree of conservation in vertebrate
species, implying the significance of these peptides in living organisms (Table 1). Ghrelin is still a ‘‘work-in-progress’’ neuropeptide when it comes to which receptors it activates. In contrast to NPY, somatostatin, and galanin, which have a number of known receptors, for ghrelin GHSR1a is the only known receptor, although ghrelin is known to act on nonGHSR1a receptors (Camina, 2006). It is of interest that this sole known receptor of ghrelin has special characteristics relative to the receptors of other neuropeptides in that it signals ligand independently with about 50% of maximal activity, and thus is constitutively active. The concept of ghrelin being an anticonvulsant is still in its initial phase, and thus it would be interesting to further contrast ghrelin with these three well-established anticonvulsant neuropeptides and elaborate regarding BBB passage, onset and duration of anticonvulsant actions, effects on LTP and memory, possible implications with respect to the ketogenic diet, and possible side effects. Transportation across the BBB A clear benefit of ghrelin has over the selected neuropeptides is the ease with which it crosses BBB (Banks et al., 2002); lack of BBB permeability to many substances can be a major obstacle to the delivery of drugs to the CNS. Analogs that cross the BBB are being developed for NPY, somatostatin, and galanin. Recently an NPY analog containing the lipidization-cationization motif, NPY-BBB2, was designed that could efficiently cross the BBB (Green et al., 2010). NPY-BBB2 administered intraperitoneally showed pronounced anticonvulsant effects in the 6-Hz mouse model of epilepsy. Somatostatin analogs that efficiently cross the BBB are yet to be discovered. As for galanin, two nonpeptide low-molecular-weight galanin receptor ligands, galnon and galmic, were designed and synthesized. Both galnon and galmic are able to cross the BBB, act as agonists with micromolar affinity toward the galanin receptors, and show anticonvulsant properties (Saar et al., 2002; Bartfai et al., 2004). Recently a nonpeptide-type positive allosteric
Table 3. Ghrelin in comparison with NPY, somatostatin, and galanin Ghrelin
Neuropeptide Y 36 Inhibits Crosses at slow rate in a nonsaturable manner
14/28 Inhibits Unable to cross BBB
29/30 Inhibits Unable to cross BBB
Receptors
28 Inhibits Easily crosses via saturable mechanisms GHSR1a
Y1, Y2, Y4, Y5, Y6, and Y7
GAL 1, 2, 3
Receptor type Receptor/s present in hippocampus
GPCR GHSR1a
GPCR Y1, Y2, Y5
Receptor/s involved in epilepsy
GHSR1a?
Release during epileptogenesis Food intake
fl Stimulates
Y2 Y1, Y5? › Stimulates
sst1–sst5, with two isoforms of sst2 (sst2A and sst2B) GPCR sst1–sst4. Presence of sst5 still debated sst2 (rats) and sst4 (mice) most effective › Inhibits
Number of residues in peptide Epilepsy Transportation across BBB
Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
Somatostatin
Galanin
GPCR GAL1, GAL2 GAL1, GAL2 › Stimulates
591 Ghrelin and Epilepsy modulator of the GAL2 receptor CYM2503, which is also CNS-penetrating, exhibited potent anticonvulsant actions following systemic administration (Lu et al., 2010). This is of interest because, as a result to their mode of action, allosteric modulators may comprise a gentler side-effect profile than tonically active exogenous agonists. Another interesting aspect of ghrelin is that it was shown to prevent the disruption of the BBB following traumatic brain injury (Lopez et al., 2012), the latter being a major risk factor for focal epilepsy. Onset and duration of anticonvulsant actions In a 2002 study, Mazarati & Wasterlain compared the anticonvulsant actions of NPY, somatostatin, and galanin in a model of self-sustaining status epilepticus. Intrahippocampal administration of somatostatin and NPY led to the immediate attenuation of both spikes and seizures that lasted for 2–3 h, followed by a second decline in seizure frequency resulting in the disappearance of seizures after 8 h of seizure induction. Galanin on the other hand suppressed spikes within 10 min and seizures within 20 min of administration, rendering it a strong anticonvulsant neuropeptide with irreversible suppression of both seizures and spikes. To date ghrelin has not been administered intrahippocampally in epilepsy models. Nevertheless in order to accurately compare ghrelin’s seizure inhibition properties against NPY, somatostatin, and galanin, ghrelin together with the aforementioned neuropeptides should be tested in the same epilepsy model. Comparison experiments like these are helpful in understanding better the individual neuropeptide profiles in relation to onset of action, anticonvulsant potential, and duration of action in different models of epilepsy. LTP and memory As mentioned earlier in this review, ghrelin was shown to promote LTP in the hippocampus as well as memory formation. Somatostatin is also known to possess similar characteristics as ghrelin, whereas both NPY and galanin do not (Badie-Mahdavi et al., 2005; Kluge et al., 2008; Sorensen et al., 2008; Ogren et al., 2010). Central administration of somatostatin improved memory and attenuated electroconvulsive shock-induced amnesia in rats (Vecsei et al., 1983; Lamirault et al., 2001), and recently it was found that deletion of the somatostatin gene markedly reduces LTP in hippocampal CA1 (Kluge et al., 2008). The role NPY plays in memory and learning is rather limited. It was shown that recombinant adeno-associated viral (rAAV) vector-induced NPY overexpression in the rat hippocampus decreased hippocampal activity-dependent plasticity (LTP) in excitatory synapses and delayed learning (Sorensen et al., 2008). In 2010, the group of Vezzani presented their data on a more efficient rAAV-NPY vector, the rAAV1-NPY, which mediated powerful anticonvulsant actions without alterations in learning and memory (Noe et al., 2010). Galanin significantly inhibits LTP in the hippocampal CA1 (Coumis &
Davies, 2002) and dentate gyrus regions of rodents (Badie-Mahdavi et al., 2005). Galnon and galmic also showed LTP inhibition effects in hippocampal slices (Badie-Mahdavi et al., 2005). Intrahippocampal and i.c.v. administration of galanin impaired learning and memory in rodents (Ogren et al., 2010). This means that the hippocampal actions on LTP by ghrelin and somatostatin, which are excitatory in nature, do not correlate directly with the peptides’ ability to prevent epileptic seizures. The combined beneficial effects on both seizures and memory tempt us to suggest that ghrelin and somatostatin might be promising candidates for future clinical applications. Ketogenic diet The ketogenic diet is a high-fat, low-protein, low-carbohydrate diet that is used primarily to treat children whose epilepsy is refractory to conventional AEDs. This diet forces the body to burn fats rather than carbohydrates, resulting in the enhanced production of ketone bodies. Various hypotheses have been put forward in order to identify why this diet is so effective. A recent review from this journal discussed the possibilities of the involvement of the norepinephrine system and the potential contribution of galanin and NPY (Weinshenker, 2008). Apart from their anticonvulsant properties, both galanin and NPY are orexigenic neuropeptides just like ghrelin. Experiments in rodents showed that the ketogenic diet did not alter galanin and NPY mRNA expression (Tabb et al., 2004) or plasma ghrelin levels (Honors et al., 2009). Weinshenker reflects that the absence of neuropeptide changes does not necessarily rule out a contribution of orexigenic neuropeptides to the anticonvulsant effect of the KD. One possibility could be that the effect of KD on these neuropeptides becomes evident following seizure activity. This is where ghrelin differs from galanin and NPY because, although ghrelin levels appear to decrease following seizure activity, the opposite takes place for NPY and galanin. Therefore, it is of interest that these orexigenic neuropeptides are further investigated to elucidate whether or not there is indeed a link between their anticonvulsant potential and the ketogenic diet. As for the anorexigenic somatostatin, a decrease in somatostatin levels was observed in a low-carbohydrate/normal-protein diet, with no changes with a low-carbohydrate/low-protein diet (Bielohuby et al., 2011). Possible side effects No drug is devoid of undesired effects. Being orexigenic, an expected side effect of ghrelin would undeniably be weight gain. This side effect is, however, also shared by the orexigenic neuropeptides galanin and NPY. Being of a pleiotropic nature, ghrelin will certainly have an effect on other biologic systems in the body. For instance, ghrelin would not be the ideal pharmacologic treatment for female patients of reproductive age, since it has been shown that Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
592 J. Portelli et al. ghrelin has a potent inhibitory effect on reproduction (TenaSempere, 2008). As with other neuropeptides, ghrelin mimetics with enhanced and more specific properties are being produced and tested with the hope of reducing the list of undesired effects.
How Can We Understand Better Ghrelin’s Role in Epilepsy? What is clear is that up to now the majority of rodent data are consistent in showing that ghrelin is anticonvulsant. However, there is still a long way to go in order to determine ghrelin’s exact role in epilepsy. The following are a few additional points that should be investigated in order to start understanding better the link between ghrelin and GHSR1a in epilepsy. What role does the GHSR1a play in epilepsy? To date the role of GHSR1a in epilepsy has not been investigated appropriately. What is known to date is that GHSR1a mRNA and protein levels showed no significant changes at 24 h after pilocarpine-induced seizures in rodents when compared to the control group (Xu et al., 2009). This group also noted that ghrelin treatment did not change this outcome on expression. It is becoming increasingly recognized that GHSR1a is not the sole receptor that ghrelin binds to (Camina, 2006). For instance there is ample proof that in chondrocytes (Caminos et al., 2005) and cardiomyocytes (Iglesias et al., 2004), ghrelin acts on receptors different from GHSR1a. Currently there is one study, by the group of Lee, who state that the low-affinity GHSR1a antagonist D-Lys3-GHRP6 blocked ghrelin’s anticonvulsant effect while having no effect on seizures when administered on its own (Lee et al., 2010). One way of verifying whether ghrelin requires GHSR1a for its anticonvulsant properties as well as investigating GHSR1a’s role in epilepsy is by using GHSR1a KO mice and high-affinity antagonists. Knowing the receptor ghrelin acts on for its anticonvulsant property is of essence in trying to decipher ghrelin’s mechanism of action in epilepsy. To what extent is the constitutive activity of the GHSR1a of importance in epilepsy? It is of great importance to understand well this specific characteristic of GHSR1a when it comes to epilepsy. In 2003, the laboratory of Holst found that GHSR1a is highly constitutively active by measuring inositol phosphate turnover and by using a reporter assay for transcriptional activity controlled by cAMP-responsive elements in COS-7 or human embryonic kidney 293 cells (Holst et al., 2003). Because GHSR1a signals with about 50% of maximal activity in the absence of its peptide ligands, it is tempting to suggest that one should in fact be looking at inverse agonists instead of neutral antagonists to block the effect of Epilepsia, 53(4):585–595, 2012 doi: 10.1111/j.1528-1167.2012.03423.x
GHSR1a. The same group presented [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P as a potent and highly efficacious inverse agonist for GHSR1a and characterized it in vivo (Holst et al., 2003; Petersen et al., 2009). Consequently, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P significantly decreased food intake and body weight when compared to rats treated with an inactive control peptide (Petersen et al., 2009). Pantel et al. (2006) highlighted the importance of the constitutive activity of GHSR1a in humans. They showed that a missense mutation of GHSR1a, which resulted in the loss of the receptor’s constitutive activity while retaining its ability to respond to ghrelininduced signaling, resulted in familial short stature. First, one should determine whether the known GHSR1a antagonists are indeed neutral antagonists or else have inverse agonist properties. It would be interesting to determine the properties of GHSR1a inverse agonists in rodent models of epilepsy. The use of mice genetically knocked out of the GHSR1a is of essence, since it gives a clear picture of whether the absence of GHSR1a provokes or inhibits seizures. In view of the rodent data available to date one would expect GHSR1a KO mice to have lower seizure thresholds than their WT littermates, as well as proconvulsant actions following pharmacologic administration of GHSR1a inverse agonists in rodent models of epilepsy. Is GHSR1b of importance in epilepsy? For years it was believed that GHSR1b was inactive; however, this notion was questionable since this isoform is spread widely in different tissues of the body. Nowadays it is thought to play a significant role in modulating GHSR1a and other GPCRs through GPCR homo- and/or heterodimerization, and it is thought to be a negative regulator of GHSR1a (Chu et al., 2007; Leung et al., 2007). Assuming that GHSR1a plays a role in epilepsy, to what extent would the importance of GHSR1b in epilepsy be? Is des-acyl ghrelin also involved in epilepsy? Ghrelin in its acylated form comprises only a small percentage of the circulating ghrelin, since 80–90% is not acylated (des-acyl ghrelin) (Kojima & Kangawa, 2005). Des-acyl ghrelin has a tissue-level distribution pattern similar to that of ghrelin, which changes during development (Nishi et al., 2005), and both forms share or oppose several functions (Soares & Leite-Moreira, 2008). A very recent study identified that des-acyl ghrelin has specific receptors in cardiomyocytes cell surface, which are distinct from those to which ghrelin binds (Lear et al., 2010). Other studies have also found non-GHSR1a des-acyl ghrelin binding site that could also bind ghrelin (Camina, 2006; Filigheddu et al., 2007). Currently there is only one study that indicates that des-acyl ghrelin may be beneficial in limbic seizures (Biagini et al., 2011). It is, therefore, necessary to study desacyl ghrelin in different epilepsy models, and if it indeed
593 Ghrelin and Epilepsy proves to show seizure protection abilities, to try to identify the receptor it acts on as well as its mechanism of action.
Concluding Remarks The discovery of ghrelin, which affects several physiologic processes and also epilepsy, has without a doubt stirred a lot of excitement in the scientific community. We feel that ghrelin ligands have great clinical potential in the field of epilepsy; however, it is important that the mechanism of anticonvulsant action exerted by ghrelin is established. GHSR1a happens to be a captivating constitutively active complex receptor, and a better understanding of ghrelin, GHSR1a, and its related receptors in vivo needs to be achieved to further clarify the role ghrelin plays in epileptic mechanisms.
Acknowledgments We thank the Vrije Universiteit Brussel and the FWO Vlaanderen (G.0163.10N) for financial support. Jeanelle Portelli is a doctoral research fellow supported by the Research Council (Onderzoeksraad – OZR1443) of the Vrije Universiteit Brussel.
Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
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