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NEUROPHARMACOLOGY
Department of Pharmaceutical Sciences and the Neurosciences Program, University of Connecticut, Storrs, Connecticut (D.A.K., S.L.K., B.A.B.); and Center for Drug Discovery, Northeastern University, Boston, Massachusetts (D.A.K., J.T.W., J.S.W., S.L., A.M., B.A.B.)
Received January 19, 2007; accepted May 30, 2007.
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Abstract |
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; Karanian and Bahr, 2006). The endocannabinoid system has been identified as a potential target for the treatment of several disorders of the central nervous system (Lastres-Becker et al., 2002; Maccarrone et al., 2003; Marsicano et al., 2003; Ramírez et al., 2005), including epilepsy and excitotoxicity (Marsicano et al., 2003; Karanian et al., 2005b; Monory et al., 2006). Endocannabinoid responses indeed have been linked to protection against a variety of neuropathogenic insults.
The neuroprotective effects elicited by the endocannabinoid system may involve modulation of ion channels (Deadwyler et al., 1993; Shen and Thayer, 1996, 1998; Mu et al., 1999) as well as signaling pathways elicited by cannabinoid CB1 receptors. The CB1 receptor, which recognizes the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol, is linked to several prosurvival signaling cascades, including phosphatidylinositol-3 kinase, protein kinase B/Akt, brain-derived neurotrophic factor, focal adhesion kinase, mitogen-activated protein kinase (MAPK), and its subtype extracellular signal regulated-kinase (ERK) (Bonni et al., 1999; Derkinderen et al., 2001, 2003; Galve-Roperh et al., 2002; Gómez del Pulgar et al., 2002; Molina-Holgado et al., 2002; Khaspekov et al., 2004; Karanian et al., 2005a,b). Disruption of CB1 receptor signaling has been shown to increase excitotoxic vulnerability and seizure susceptibility, as well as attenuate neuronal maintenance (Wallace et al., 2002, 2003; Clement et al., 2003; Marsicano et al., 2003; Khaspekov et al., 2004; Karanian et al., 2005a). These studies indicate the importance of endocannabinoid responses in cell survival and the viability of neuronal networks.
The endocannabinoid system comprises at least two types of cannabinoid receptors, an endocannabinoid transport system and endocannabinoid-degrading enzymes. Cannabinergic inactivation involves internalization of endocannabinoids by the highly selective carrier-mediated transport system (Di Marzo et al., 1994; Beltramo et al., 1997; Piomelli et al., 1999; Fegley et al., 2004), as well as enzymatic degradation. Fatty acid amide hydrolase (FAAH), which is distributed throughout the brain, is thought to be the primary mediator for the hydrolysis of released endocannabinoids (Cravatt et al., 1996, 2001; Egertová et al., 2003). Enhancement of endocannabinoid responses can be achieved with disruption of FAAH activity (Cravatt et al., 2001; Kathuria et al., 2003), as well as through inhibition of the transporter (Giuffrida et al., 2000; Fegley et al., 2004; Karanian et al., 2005b). Thus, cannabinergic tone can be enhanced by blocking the inactivation of endogenous ligands.
Here, we tested whether efficient blockage of endocannabinoid inactivation with the irreversible FAAH inhibitor AM374 elevates AEA in the brain and leads to a level of CB1 signaling that is sufficient to protect against excitotoxic damage. Modulation of the endocannabinoid system with AM374 was evaluated for neuroprotection against 1) kainate-induced seizures, 2) molecular and cellular indicators of excitotoxic pathology, and 3) excitotoxic behavioral impairments. The results indicate that enhancement of endocannabinoid responses through FAAH inhibition reduces seizure activity and the seizure-induced brain damage.
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Materials and Methods |
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). The animals were handled daily for 3 to 5 days to facilitate acclimation before behavioral testing. Experiments were initiated at postnatal days 21 to 24.
Chemicals and Antibodies. The FAAH inhibitor AM374 and CB1 antagonist AM251 was synthesized through an established route (Gatley et al., 1996; Deutsch et al., 1997). Kainic acid (KA) was obtained from Tocris Cookson Inc. (Ellisville, MO). Affinity-purified antibodies to GluR2/3 (Bahr et al., 1996) and to the calpain-mediated spectrin fragment BDP were used as described previously (Karanian et al., 2005b). Other antibodies utilized included those against the active form of ERK and total ERK (Cell Signaling, Beverly, MA), synapsin II (Calbiochem, San Diego, CA), and actin (Sigma-Aldrich, St. Louis, MO).
Endocannabinoid Detection. After systemic injection of AM374 or vehicle consisting of phosphate-buffered saline with dimethyl sulfoxide, brain regions were rapidly dissected, dropped into liquid nitrogen, and subjected to a methanol/chloroform extraction procedure. Chromatographic separation was achieved using an Agilent Zorbax SB-CN column (2.1 x 50 mm) on a Finnigan TSQ Quantum Ultra triple quad mass spectrometer (Thermo Electron Corporation, Waltham, MA) with an Agilent 1100 HPLC on the front end (Agilent Technologies, Palo Alto, CA). The mobile phase consisted of 10 mM ammonium acetate, pH 7.3, and methanol in the following gradient; initial conditions were held at 10% for 1 min, increased linearly from 60 to 75% for 1.5 to 10 min, and then increased to 95% in 0.5 min (flow rate, 0.5 ml/min); the autosampler was kept at 4°C to prevent analyte degradation. Eluted peaks were ionized via atmospheric pressure chemical ionization. Deuterated AEA from 0 to 500 pg/µl were used for the standard curve, and levels in brain per gram of tissue were determined (correlation coefficient, r = 0.99).
Activation of ERK. Sprague-Dawley rats were administered an i.p. injection of vehicle or 8 mg/kg AM374. The animals were sacrificed 15 to 90 min postinjection, and brain tissue was rapidly dissected. The hippocampus, frontal cortex, and neocortex samples were homogenized in lysis buffer consisting of 15 mM HEPES, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, and the protease inhibitor cocktail. Protein content was determined, and equal protein aliquots were assessed by immunoblot for active pERK2 with antibodies specific for mitogen-activated protein kinase kinase-dependent phosphorylation sites in the catalytic core of ERK2, as well as total ERK as described previously (Bahr et al., 2002; Karanian et al., 2005a).
KA-Induced Seizures and Subsequent Drug Administration. Sprague-Dawley rats were injected first with phosphate-buffered saline (n = 17) or 10 mg/kg KA (n = 21). Immediately after the KA injection, the rats were administered a subsequent i.p. injection with either vehicle or 1 to 8 mg/kg AM374. Following the injections, animals were monitored for seizure activity; 24 to 48 h postinjection, animals were assessed for behavioral changes; and at 48 h, postinjection animals were sacrificed for immunoblot and histological analysis as described. A subset of animals assessed at 48 h postinjection for molecular and cellular changes were administered 3 mg/kg AM251 30 min before receiving the excitotoxin and AM374.
Seizure Scoring. Animals were observed for 4 h after injections. Seizures were counted and scored for each hour postinjection. In brief, blinded raters continuously observed the animals throughout the 4-h period. The seizure counts and score were recorded every 15 min, representative of the seizure expression during that period. The seizure rating scale consisted of the following six stages: stage 0, normal behavior; stage 1, freezing, staring, mouth, or facial movements; stage 2, rigid posture, head nodding, or isolated twitches; stage 3, tail extension, unilateral-bilateral forelimb clonus, or repetitive scratching; stage 4, rearing with one or both forepaws extended; stage 5, clonic seizures with loss of posture, jumping, and falling; and stage 6, severe tonic seizures.
After 48 h postinjection, brains were rapidly removed with ice-cold homogenization buffer consisting of 0.32 M sucrose, 5 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.6 µM okadaic acid, 50 nM calyculin A, and a protease inhibitor cocktail containing 4-(2-amino-ethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. The hippocampal tissue was homogenized in lysis buffer as described previously (Bahr et al., 2002), and protein content was determined and assessed for cytoskeletal and synaptic markers.
Immunoblot Analysis. Protein content was determined in the homogenized hippocampal samples with a bovine serum albumin standard, and equal aliquots of the samples were denatured in SDS at 100°C. The samples were then separated by SDS-polyacrylamide gel electrophoresis and blotted to nitrocellulose. Immunodetection was achieved by incubating blots overnight at 4°C with separate antibodies to the active form of ERK and total ERK, calpain-mediated spectrin breakdown product, synapsin II, and the 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR2/3. Blots were routinely stained for a protein load control (e.g., actin). Anti-IgG-alkaline phosphatase conjugates were used for secondary antibody incubation. Development of immunoreactive species used the 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium substrate system and was terminated before maximal intensity to avoid saturation. Integrated optical density of the bands was determined at high resolution with BIOQUANT software (R and M Biometrics, Nashville, TN).
Histology. Brain tissue was fixed in paraformaldehyde for 24 h and then paraffin-embedded. The paraffin blocks were sectioned at 5-µm thickness and mounted on slides. The embedded tissue was heated at 60°F for 30 min, dehydrated through ethanol solutions, and stained with hematoxylin and eosin.
Behavioral Testing. Animals were assessed for excitotoxic-induced behavioral changes 24 to 48 h postinjection. In the first behavioral test, animals were placed with front paws over a bar and hind paws touching surface to measure time before movement. Time to front paw movement was recorded for a maximum of 30 s. The second behavioral test involved balance on a suspended beam as modified from Hamm et al. (1994). The animals were placed on a beam (1.2-cm diameter) suspended 15 cm above a padded surface and assessed for time to fall with a maximal time of 30 s. A Rotorod paradigm was used for the third behavioral test to assess motor coordination. As modified from previously published articles (Hamm et al., 1994; Kábová et al., 1999), animals received 10 consecutive training sessions on the rotating rod (10 rpm). After the final training, the animals were allowed to rest and then were tested for the ability to maintain balance and coordination on the Rotorod for a maximum of 30 s. A subset of animals was monitored for locomotor activity in a novel environment. The rats were assessed for distance of exploration in an open field (106 x 54 cm) that was divided into eight segments. The animals were placed in the center of the open field and monitored for the total number of segments crossed during the 5-min session.
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; Karanian et al., 2005a,b). The representative blots in Fig. 2A show an increased level of the phosphorylated active form of the ERK2 isoform (pERK2) within 15 min after AM374 administration. The drug resulted in the activation of ERK2 in several brain regions, including the hippocampus, frontal cortex, and neocortex (Fig. 2B). The most robust pERK2 activation was evident at 90 min postinjection for each brain region. AM374 increased pERK2 activation above basal levels by 400% in the hippocampus, 650% in the frontal cortex, and 284% in the neocortex. As a control, total ERK levels were found not to change with drug treatment across the different time points. Taken together, these results indicate that AM374 crosses the blood-brain barrier and enhances AEA levels to promote cannabinergic signaling.
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Figure 3B shows KA-induced seizure scores across the postinsult rating period, data for each hour normalized to the KA-only animals. AM374-induced protection was significant in the 1st h of seizure induction, and the protection progressively improved in subsequent hours. Compared with KA rats that did not receive drug, AM374 reduced seizure severity by 47% in h 1, by 44% in h 2, by 70% in h 3, and by 86% in h 4. AM374 not only reduced the overall seizure severity but also appeared to accelerate the rate of seizure decay. Thus, disruption of FAAH activity with the irreversible FAAH inhibitor effectively reduces excitotoxin-induced seizure events.
Neuroprotection. The KA and KA plus AM374 groups were also assessed for molecular and cellular indicators of excitotoxicity. At 48 h postinjection, brains were rapidly dissected under ice-cold conditions, and hippocampal tissue was assessed by immunoblot with specific antibodies. The hippocampus from KA-exposed animals exhibited a pronounced level of calpain-mediated cytoskeletal breakdown (BDP) as well as loss of synaptic markers synapsin II and GluR2/3 (Fig. 4A). Note that cytoskeletal BDP is often associated with a loss of synaptic and dendritic integrity (Neumar et al., 2001; Bahr et al., 2002; Karanian et al., 2005b), and we have previously reported that enhancement of endocannabinoid responses protects against such pathogenic manifestations (Karanian et al., 2005b). When AM374 was injected immediately following the KA insult, spectrin BDP measured 48 h later was reduced by 89% (Fig. 4B; ANOVA, p < 0.01). Thus, the FAAH inhibitor provides nearly complete cytoskeletal protection from KA-induced excitotoxicity.
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To assess cellular changes, brains from the different treatment groups were rapidly dissected 48 h postinsult and then fixed and sectioned for staining with hematoxylin and eosin. As shown in photomicrographs of CA1 of hippocampus, exposure to the excitotoxin resulted in neuronal compromise. Compared with control tissue (Fig. 6B), the KA insult dramatically reduced viable neurons and induced pyknotic changes in the pyramidal subfield (Fig. 6C). AM374 promoted cell survival as confirmed by the absence of neuronal loss and pyknotic effects in tissue from an excitotoxic animal treated with the FAAH inhibitor (Fig. 6D). As was the case for cytoskeletal and synaptic protection, pretreatment with AM251 prevented the effect of AM374 on the cellular protection (Fig. 6E). FAAH inhibition thus protects against excitotoxin-induced neuronal compromise by acting through the CB1 receptor.
Functional Protection. Three behavioral paradigms were employed to test the irreversible FAAH inhibitor for functional protection. The first behavioral test assessed onset of front paw movement when placed on a raised bar as a measure of the animal's coordination to negotiate the raised position. As shown in Fig. 7A, KA-induced neurodegeneration was associated with a marked delay in initial movement assessed 24 h postinsult over the basal movement exhibited by vehicle-injected control animals. Initiation of activity was returned to near-control levels when endocannabinoid inactivation was inhibited by injection of AM374 immediately following the excitotoxin exposure (Fig. 7A; ANOVA, p < 0.001; post hoc test compared with insult-only data, p < 0.01). Note that the KA alteration was not due to gross impairment in motor ability because there was no change across treatment groups in explorative distance when placed in a novel open field (Fig. 7B).
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; Kábová et al., 1999). At 48 h postinjection, rats were trained on the Rotorod and subsequently tested for their ability to maintain themselves for a continuous period on the 10-rpm rotating rod. The excitotoxic damage induced by KA exposure resulted in a marked reduction in Rotorod performance compared with control animals (Fig. 8B). AM374 protected against the excitotoxin-induced impairment as determined by analysis of variance (p < 0.01), thus indicating improved motor coordination. The assessment of selective motor disturbances further supports that the irreversible FAAH inhibitor reduces excitotoxic brain defects. Taken together, these results demonstrate that enhancement of endocannabinoid responses protects against excitotoxic seizures and seizure-induced neuronal damage.
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), whereas their effect on endogenous AEA concentrations indirectly promotes cannabinergic responses. AM374 has a similar selectivity for FAAH compared with monoglyceride lipase, which hydrolyzes 2-arachidonoyl glycerol (Dinh et al., 2002). Disrupting endocannabinoid responses alters synaptic integrity, increases excitotoxic vulnerability, and attenuates survival responses (Parmentier-Batteur et al., 2002; Marsicano et al., 2003; Khaspekov et al., 2004; Karanian et al., 2005a,b). In contrast, the current study demonstrates that enhancing endocannabinoid levels through the selective inhibition of FAAH protects against KA-induced seizures and excitotoxic neurodegeneration. Moreover, modulating the endocannabinoid system in this manner also ameliorated excitotoxic brain defects evident in behavioral tests.
AM374-mediated enhancement of endocannabinoid responses in our study was associated with reduced seizures in the KA rat. The KA excitotoxin is well known to induce seizures and pathology characteristic of epilepsy. We showed that FAAH inhibition provides a dose-dependent as well as accelerated rate of seizure recovery. The protection against seizures was evident throughout the seizure-scoring period, suggesting that the drug has an extended effect on reducing excitotoxic progression. Note that although FAAH knockout mice were found to exhibit susceptibility to spastic and proconvulsant activity (Cravatt et al., 2001; Clement et al., 2003), dosing with the potent AM374 compound facilitated a controlled modulation of FAAH to promote protective cannabinergic signals. In relation to the present results, inhibiting endocannabinoid transport has been shown to reduce motor hyperactivity and seizure severity (Lastres-Becker et al., 2002; Marsicano et al., 2003), and several types of neuroprotection have been described with agonists that activate CB1 responses (see Bahr et al., 2006). When endocannabinoid signaling is disrupted through genetic or pharmacological means, the resultant proconvulsant conditions can lead to neuronal compromise (Wallace et al., 2002, 2003; Marsicano et al., 2003; Monory et al., 2006).
In the KA model, we also tested for protection against molecular and cellular consequences ascribed to epileptiform-type excitotoxic events. AM374 prevented KA-induced cytoskeletal damage and synaptic decline in the hippocampus. The cytoskeletal damage was assessed by measuring calpain-mediated BDP, a sensitive precursor to synaptic compromise and subsequent neuronal pathology in in vitro and in vivo models (see Bahr et al., 2002; Karanian et al., 2005b). As in such studies, pharmacological reduction of excitotoxic cytoskeletal damage was associated with enhanced survival of important neuronal fields. Both indications of protection mediated by AM374 were prevented by the CB1 antagonist AM251. Corresponding with the cytoskeletal protection, disruption of endocannabinoid inactivation also provided nearly complete protection of pre- and postsynaptic protein expression. As in the case of the cytoskeletal and cellular protection, CB1 antagonism also prevented the synaptic preservation, indicating that the protective effects of AM374 are mediated by CB1 receptors. It should be pointed out that KA-induced seizure severity correlates with synaptic marker decline, probably leading to the loss of synapse function. The reduction of the excitotoxic cascade through governed excitability and preserved integrity of synapses by AM374 would perhaps safeguard those synaptic signals that are important for neuronal maintenance (see Hayashi et al., 1999; McKinney et al., 1999; Bahr et al., 2002). The data support the idea that FAAH inhibition provides cellular protection against epileptiform excitotoxicity.
In addition to the cytoskeletal, synaptic, and cellular protection, AM374 effectively ameliorated behavioral indications of excitotoxic brain damage. The KA insult caused marked impairment of balance and coordination as indicated by reduced performance on three paradigms. The behavioral alterations were significantly improved when the FAAH inhibitor was administered immediately after the KA injection. The data strongly suggest preservation of brain function because the level of KA used did not affect gross motor ability as indicated by normal explorative motor behavior. Interestingly, the KA plus AM374 rats also did not exhibit deficits in explorative movement, suggesting that enhancement or maintenance of KA-induced AEA levels does not produce adverse motor effects. Others have shown that the AEA transport inhibitor LY2183240 elevates the endocannabinoid 5-fold in the brain, resulting in behaviorally relevant cannabimimetic responses in the absence of adverse effects on motor function (Moore et al., 2005). Our functional protection results correspond with previous work that showed that inhibition of endocannabinoid inactivation attenuates excitotoxin-induced behavioral defects (Karanian et al., 2005b). FAAH inhibition has also been reported to reduce muscle spasticity in a multiple sclerosis model (Baker et al., 2001). Together, the findings indicate that endocannabinoid modulation via FAAH inhibition protects against excitotoxicity and preserves brain function.
The present findings further support that the endocannabinoid system provides an on-demand response to excitotoxic insults as previously implicated (see Marsicano et al., 2003; Karanian et al., 2005b). Excitotoxins, including KA, have been reported to selectively increase AEA levels in the brain by 3- to 13-fold, prompting the idea that CB1 responses facilitate endogenous protection (Hansen et al., 2001; Marsicano et al., 2003). The increase in the diffusible messenger after systemic KA administration in mice was transient, peaking after 20 min and returning to normal levels after 60 min. Controlled modulation of FAAH may have prolonged such transient responses because AM374 was still effective 3 h post-KA, increasing AEA 3 to 6-fold. Enhancement of the endocannabinoid system reduced KA-induced seizure severity, perhaps by reducing intracellular calcium via cannabinergic actions on voltage-gated calcium channels and modulation of potassium channels through inhibition of adenylyl cyclase (Deadwyler et al., 1993; Shen and Thayer, 1996, 1998; Mu et al., 1999), as well as by eliciting nonspecific and synapse-specific depression of excitatory circuits (Shen et al., 1996; Kim and Thayer, 2000; Gerdeman and Lovinger, 2001; Singla et al., 2007). Induced calcium regulation is supported by the fact that FAAH inhibition markedly decreased calcium-dependent activation events of calpain that produce cytoskeletal fragments. The reduction in calcium would govern neuronal excitability, slow excitotoxic progression that expands out from zones expressing seizure activity, and attenuate calpain and other forms of pathogenesis that lead to cellular and functional compromise. Indeed, AM374 protected against KA-induced behavior defects, and previous work reported that endocannabinoid enhancement produces a neuroprotective correlation between reduced cytoskeletal breakdown mediated by calpain and improved performance in a fear-conditioning paradigm (Karanian et al., 2005b). In addition to calcium regulatory pathways, enhanced cannabinergic responses during the postinsult recovery period may also entail MAPK and other prosurvival signal transducers. AM374 seems to have more than a transient effect on the activation of the ERK/MAPK pathway, a pathway found to be through the upstream activator mitogen-activated protein kinase kinase, and mediated by CB1 receptors (Derkinderen et al., 2003; Karanian et al., 2005a,b). The additional CB1 events that may be involved cover a wide range of prosurvival signal transduction elements, including phosphatidylinositol-3 kinase/Akt, brain-derived neurotrophic factor, and focal adhesion kinase (see Hayashi et al., 1999; McKinney et al., 1999; Nagayama et al., 1999; Panikashvili et al., 2001; Gómez del Pulgar et al., 2002; Molina-Holgado et al., 2002; Maccarrone et al., 2003; Marsicano et al., 2003; Khaspekov et al., 2004; Karanian et al., 2005a), possibly explaining the broad protective effects of the endocannabinoid system across diverse disease states.
Endocannabinoids are elevated following seizure induction and are possibly synthesized and released on demand to prevent excitotoxic progression (Wallace et al., 2002, 2003; Marsicano et al., 2003). Accordingly, pharmacological modulation of endocannabinoid tone has become of great interest, and several reports indicate the neuroprotective effects of inhibiting FAAH activity (for recent review, see Bahr et al., 2006). The present report is consistent with such results, suggesting that compensatory signaling can be positively modulated through the inhibition of endocannabinoid hydrolysis with AM374. The endocannabinoid system is probably involved in a negative feedback loop to inhibit neuronal overexcitation, as well as a positive feedback loop of cell maintenance signaling. To summarize, enhancement of endocannabinoid responses through inhibition of FAAH is sufficient to produce molecular, cellular, and functional protection in a model of epileptiform excitotoxicity. AM374 seems to cross the blood-brain barrier and effectively enhance endocannabinoid signaling. Such indirect modulation of endocannabinoid responses promotes recovery from seizure events and improves brain function. Thus, controlled FAAH inhibition poses a therapeutic avenue for epileptiform excitotoxicity.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CB1, cannabinoid CB1 receptor; AEA, anandamide; MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated-kinase; FAAH, fatty acid amide hydrolase; AM374, palmitylsulfonyl fluoride; AM251, N-(piperidin-1-yl)-5-(4-iodophonyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; KA, kainic acid; BDP, spectrin breakdown product; E-64, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide; ANOVA, analysis of variance; LY2183240, 5-biphenyl-4-ylmethyltetrazole-1-carboxylic acid dimethylamide.
Address correspondence to: Dr. David A. Karanian, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092. E-mail: david.karanian{at}uconn.edu
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