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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Neuropharmacology. 2021 Nov 22;205:108897. doi: 10.1016/j.neuropharm.2021.108897

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome

Lyndsey L Anderson 1,2,3, Peter T Doohan 1,2,3, Nicole A Hawkins 4, Dilara Bahceci 1,2,3, Ganesh A Thakur 5, Jennifer A Kearney 4, Jonathon C Arnold 1,2,3
PMCID: PMC9514665  NIHMSID: NIHMS1837540  PMID: 34822817

Abstract

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/− mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/− mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/− mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/− phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of the Scn1a+/− mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.

Keywords: endocannabinoid system, Dravet syndrome, cannabinoid receptor

1. INTRODUCTION

Epilepsy is a common neurological disease that affects approximately 50 million people worldwide (Beghi et al., 2019). While disease and injury to the CNS are known to contribute to the pathology of epilepsy, nearly two-thirds of human epilepsies are presumed to be genetic in origin, with complex inheritance patterns. Mutations in voltage-gated sodium channels are the most prevalent cause of monogenic epilepsy. Within these monogenic epilepsies, identical sodium channel mutations can result in epilepsy phenotypes with varying severity (Cetica et al., 2017; Meisler et al., 2010). These diverse epilepsy phenotypes suggest that disease severity is influenced by other factors, including genetic modifiers. Genetic modifiers are genes distinct from the primary mutation that modulate the severity of the disease phenotype (Kearney, 2011). Seizure phenotypes in mouse models of epilepsy also show variable seizure severity and can be studied to identify potential modifier genes (Hawkins et al., 2016; Miller et al., 2014). Genetic modifiers can also provide new leads on therapeutic targets, which is important since 30% of epilepsy patients are resistant to currently available treatments (Golyala and Kwan, 2017).

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. Loss-of-function mutations in SCN1A, the gene encoding Nav1.1, are present in over 80% of Dravet syndrome patients (Dravet and Oguni, 2013). Heterozygous deletion of Scn1a (Scn1a+/−) in mice mimics the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures, cognitive and behavioral deficits and reduced survival (Hawkins et al., 2017; Miller et al., 2014). Penetrance of the Dravet syndrome phenotype is highly strain-dependent. When the Scn1a+/− deletion is present on a 129S6/SvEvTac strain (129.Scn1a+/−), mice have no overt phenotype and a normal lifespan (seizure-resistant strain) (Miller et al., 2014). However, when 129.Scn1a+/− mice are crossed with C57BL/6J mice, the resultant F1 generation (F1.Scn1a+/−) exhibits a severe Dravet syndrome phenotype (seizure-susceptible strain). Previous work has identified Gabra2, the gene encoding the GABAA receptor α2 subunit, and Cacna1g, the gene encoding Cav3.1, as genetic modifiers of the Scn1a+/− mouse model of Dravet syndrome (Calhoun et al., 2017; Hawkins et al., 2016). Since both of these genes are molecular components of neuronal signaling, it is not surprising that their expression can affect the phenotype of Scn1a+/− mice. The endocannabinoid system is another important regulator of neuronal signaling.

The endocannabinoid system consists of lipid-derived signaling molecules (endocannabinoids), cannabinoid receptors (CB1 and CB2) and the enzymes that synthesize and degrade endocannabinoids. In the CNS, the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) function as retrograde neurotransmitters that are released on-demand and activate presynaptic cannabinoid receptors, which inhibit presynaptic neurotransmitter release (Bisogno et al., 1999). The phytocannabinoid cannabidiol (CBD) is an FDA-approved drug to treat intractable childhood epilepsies and one of its suggested mechanisms it to enhance endocannabinoid signaling (Blair et al., 2006; Corroon and Felice, 2019; Roebuck et al., 2021; Rowley et al., 2017). However, whether an endocannabinoid system deficiency contributes to intractable childhood epilepsies such as Dravet syndrome is unknown. We sought to determine whether a deficiency in the endocannabinoid system contributes to the Dravet syndrome phenotype of Scn1a+/− mice by first comparing hippocampal CB1 receptor expression and endocannabinoid concentrations between seizure-susceptible and seizure-resistant genetic background strains. We then pharmacologically enhanced endocannabinoid tone in the Scn1a+/− mice to explore the endocannabinoid system as an anticonvulsant drug target for Dravet syndrome.

2. METHODS

2.1. Animals.

All animal care and procedures were approved by the University of Sydney Animal Ethics Committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes or the Northwestern University Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice heterozygous for Scn1a (Scn1a+/−) were generated by targeted deletion of exon 1 and maintained as a congenic line on the 129S6/SvEvTac background (129.Scn1a+/−) as described (Miller et al., 2014). For studies conducted at the University of Sydney, 129.Scn1a+/− mice were purchased from The Jackson Laboratory (stock 37107-JAX; Bar Harbor, USA). Wildtype C57BL/6J mice were bred with heterozygous 129.Scn1a+/− mice to generate F1 (129 x B6) mice. The Scn1a genotype was determined as previously described (Hawkins et al., 2017). Mice were group-housed in specific pathogen-free mouse facilities under standard conditions with ad libitum access to food and water. Gene expression studies were conducted at Northwestern University (Chicago, USA) where the mouse facility operated under a 14h light/10 h dark cycle. All other studies were conducted at the University of Sydney (Sydney, AUS) where the mouse facility operated under a 12 h light/12 h dark cycle.

2.2. Statistical analysis.

All statistical comparisons were made using GraphPad Prism 8.2 (La Jolla, CA) and p < 0.05 was considered statistically significant. Normality of each dataset was assessed using the Shapiro-Wilk test and non-parametric analysis was used where normal distribution was not observed. Homoscedasticity was assessed using the F-test or Spearman’s test depending on the dataset. In experiments comparing endocannabinoid system components overall effects were first analyzed using three-way ANOVA with between-subject factors of ‘strain’, ‘genotype’ and ‘sex’, with the exception of Cnr1 measurements where there was no ‘sex’ factor. Initial ANOVAs were followed by two-way ANOVAs split by the corresponding factor with post-hoc analysis where appropriate. For in vivo seizure experiments, no significant sex differences were observed so males and females were combined across groups. Details regarding specific statistical tests for each experiment are presented below.

2.3. Quantitative Reverse transcription droplet digital PCR (RT-ddPCR).

Transcript expression of target genes was determined using RT-ddPCR in samples previously collected and described (Hawkins et al., 2019). Briefly, cortex and hippocampi were dissected from mice at postnatal day 24 (P24) and tissue from 3–4 mice was combined into pooled samples and total RNA was isolated. We did not expect sex to affect Cnr1 expression so each RNA pool contained both male and female samples. First strand cDNA was synthesized and ddPCR was performed using ddPCR Supermix for Probes (Bio-Rad; Hercules, USA) and TaqMan Assays as previously described (Hawkins and Kearney, 2016). Taqman gene expression assays (Life Technologies) were: mouse Cnr1 (FAM-MGB-Mm01212171_s1) and Tbp (VIC-MGB-Mm00446971_m1). Relative transcript levels were expressed as a concentration ratio to Tbp. Statistical comparisons were made using two-way ANOVA (between-subjects factors: ‘strain’ and ‘genotype’).

2.4. Immunoblotting.

Mice were euthanized by cervical dislocation between 12:00–12:15, brains were snap frozen in liquid nitrogen and hippocampi were dissected at P24. Individual hippocampi were homogenized thoroughly using a glass Dounce homogenizer in a sucrose solution (0.32 M sucrose, 5 mM Tris HCl, pH 7.4, protease inhibitor). Homogenates were centrifuged at 2,000g for 10 min at 4°C. The supernatant was then centrifuged at 20,000g for 40 min at 4°C. The pellet was resuspended in a Tris-EDTA (5 mM Tris HCl, pH 8.2, 1 mM EDTA, protease inhibitor) solution, homogenized further in a glass Dounce homogenizer and centrifuged at 20,000g for 40 min at 4°C. Lastly, the pellet was resuspended in a Tris-EDTA buffer (50 mM Tris, pH 7.5, 10 mM EDTA, protease inhibitor). Protein concentrations were determined by Bradford assay prior to Western blot analysis. Membrane proteins (20 μg) were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Proteins were detected with primary antibodies directed against CB1 (rabbit; anti-CB1 polyclonal; 1:200; ab23703; Abcam; Cambridge, GBR) and β-tubulin (mouse; anti-β-tubulin monoclonal; 1:500; T5201; Sigma-Aldrich; St. Louis, USA) and/or mortalin/GRP75 (mouse; anti-mortalin/GRP75 monoclonal; 1 μg/mL NeuroMab N52A/42). Immunoreactive bands were detected with an Odyssey imager (LI-COR Biosciences; Lincoln, USA) using fluorescent secondary antibodies directed at the primary antibodies (goat:anti-rabbit 800 or goat:anti-mouse 680; 1:20,000; ThermoFischer Scientific; Waltham, USA). Densitometry was performed and band intensity was normalized to that of β-tubulin. Statistical comparisons were made using three-way ANOVA (between-subjects factors: ‘strain’, ‘genotype’ and ‘sex’) then two-way ANOVA (between-subjects factors: ‘strain’ and ‘genotype’) for genetic modifier experiments. Student’s t-test was used to compare hippocampal CB1 receptor levels in subchronically treated F1.Scn1a+/− mice.

2.5. Endocannabinoid analysis.

Whole brains were harvested between 12:00–12:15 from mice euthanized by cervical dislocation without the use of anaesthesia because brain endocannabinoid concentrations have been shown to transiently increase with anaesthesia use (Patel et al., 2003). Brains were snap frozen in liquid nitrogen and stored at −80°C until analysis. Hippocampi were dissected and 2 mL of dichloromethane and methanol (2:1) and 5 μL of an internal solution were added (consisting of 1 μg/mL d4-anandamide, 10 μg/mL d5-2-AG, 100 μg/mL d8-arachidonic acid and 100 μg/mL d8-1-stearoyl-2-arachidonoyl-sn-glygerol (SAG)). Hippocampi were homogenized thoroughly using a glass Dounce homogenizer. Homogenates were centrifuged at 20,000g for 30 mins at 4°C. The supernatant (1 mL) was transferred to a glass tube and 2 mL of dichloromethane and methanol (2:1) was added. Next, 1.5 mL of 0.88% KCl in water (w/v) was added to each sample for a two-phase liquid-liquid extraction. Phases were allowed to separate overnight at −20°C. Following aspiration of the upper aqueous layer, the bottom organic layer was transferred to a clean glass tube and dried to completeness under nitrogen gas. Samples were reconstituted in 35 μL of methanol and 15 μL of 0.1% formic acid in water for analysis. Samples remained on ice throughout the preparation process and the use of plastic consumables (with the exception of pipette tips) was avoided to maximize recovery and eliminate isomeric conversion of 2-AG to 1-AG.

Hippocampal concentrations of 2-AG, 1-AG, anandamide, SAG and arachidonic acid were assayed using a Shimadzu Nexera ultra-high-performance liquid chromatograph coupled to a Shimadzu 8040 triple quadrupole mass spectrometer (LC-MS/MS; Shimadzu Cor.; Kyoto, JPN). Samples were separated on a ZORBAX XDB-C18 column (Agilent Technologies; Santa Clara, USA) using a gradient elution. Mobile phases, 0.1% aqueous formic acid (A) and methanol (B) were delivered at a flow rate of 0.6 L/min as follows: mobile phase B was held at 70% for 0.25 mins then rapidly increased to 77% and held for 5.6 mins. Mobile phase B was then rapidly increased to 100% and held for 3.5 mins before returning to 70% to equilibrate. The mass spectrometer was operated in positive electrospray ionization mode with multiple reaction monitoring. Details regarding MS conditions and limits of quantification for each analyte are provided in Supplemental Table 1. Samples were compared to external calibration curves to quantify endocannabinoid concentrations. Normal distribution and equal variance was not observed for the 1-AG dataset so the concentrations were transformed to 1/concentration before statistical analysis. Statistical comparisons of all endocannabinoids were made using three-way ANOVA (between-subjects factors: ‘strain’, ‘genotype’ and ‘sex’). Concentrations of 2-AG were then compared using two-way ANOVA (between-subjects factors: ‘strain’ and ‘sex’).

2.6. Compounds.

ABX-1431 was purchased from WuXi App Tec (Shanghai, CHN). GAT229 was synthesized as previously described, with > 99% enantiomeric purity (Laprairie et al., 2017). For acute administration, compounds were prepared fresh on the day of the experiment as solutions in ethanol:Tween 80:saline (1:1:18). GAT229 doses of 30 mg/kg and 100 mg/kg were suspensions. Compounds were administered as an intraperitoneal (i.p.) injection in a volume of 10 mL/kg.

2.7. Pharmacokinetic analysis.

Wildtype mice (P21–28) received an i.p. injection of 5 mg/kg GAT229 or ABX-1431. At selected time points mice were anesthetized with isoflurane, whole blood was collected by cardiac puncture and brains were harvested. Plasma was isolated by centrifugation (9000 g for 10 min, 4 °C) and samples were stored at −80 °C until assayed. Compound concentrations at each time point were averaged and pharmacokinetic parameters were calculated by noncompartmental analysis as previously described (Hawkins et al., 2017).

2.8. Hyperthermia-induced seizures.

Hyperthermia-induced seizure experiments were conducted on male and female F1.Scn1a+/− mice at P14–16 as previously described (Hawkins et al., 2017). Briefly, fifteen minutes prior to the target experimental (post-dose) time point for each compound, a RET-3 rectal temperature probe was inserted and mice acclimated for 5 min. Mouse core body temperature was then elevated 0.5°C every 2 min until the onset of first clonic convulsion with loss of posture or until 42.5 °C was reached. Mice that reached 42.5°C were held at temperature for 3 minutes and were considered seizure-free if no seizure occurred during the hold. Immediately following the hyperthermia-induced seizure protocol, plasma and brain samples were isolated as described for the pharmacokinetic analysis. In a subset of the ABX-1431 experimental mice, whole brains were harvested and snap frozen in liquid nitrogen from mice euthanized by cervical dislocation without the use of anaesthesia to analyze endocannabinoid concentrations. Experimental time points were 30 min (GAT229) and 60 min (ABX-1431) based on our pharmacokinetic studies. The experimental time point for ABX-1431 was chosen as 60 min because a substantial concentration had accumulated in the brain at this time point and previous studies have shown that MAGL inhibition occurs immediately after exposure (Long et al., 2009b). Seizure threshold temperatures were compared using Mantel-Cox logrank test. Statistical comparisons of 2-AG concentrations in ABX-1431 experimental mice were made using Brown-Forsythe ANOVA to correct for unequal variance, followed by Dunnett’s T3 post hoc. Statistical comparisons of anandamide concentrations in ABX-1431 experimental mice were made using Kruskal-Wallis test.

2.9. Spontaneous seizures and survival.

Male and female F1.Scn1a+/− mice were exposed to a single hyperthermia-induced seizure event at P18 as described previously (Hawkins et al., 2017). Mice were then randomly assigned to untreated or ABX-1431 treatment groups. ABX-1431 was administered orally through supplementation in chow formulated in house using R&M Standard Diet (Specialty Feeds; Glen Forrest, AUS) irradiated powder at 350 mg ABX-1431/kg chow. Continuous video recordings were captured for 60 h and spontaneous generalized tonic-clonic seizures were quantified and scored as previously described (Hawkins et al., 2017). Mice continued treatment to P30 to monitor survival. Brains were harvested from survival mice on P31 by cervical dislocation without the use of anaesthesia and brains were flash frozen in liquid nitrogen to analyze endocannabinoid concentrations. Separate cohorts of wildtype mice treated with ABX-1431 were used to determine plasma concentrations at P21, P25 and P31. Following subchronic ABX-1431 treatment, plasma was isolated as described above within 30 min of lights on. Plasma ABX-1431 concentrations were as follows: 170 ± 19 ng/mL (P21), 394 ± 49 ng/mL (P25) and 297 ± 74 ng/mL (P31), which correspond to 3, 7 and 13 days of ABX-1431 treatment, respectively. Statistical comparisons were made using Fisher’s exact test (proportion of mice seizure-free and proportion of hindlimb seizures), Mann-Whitney test (seizure frequency) or Mantel-Cox logrank test (survival). Statistical comparisons of the 2-AG concentrations in experimental animals were made using Welch’s t-test to correct for unequal variance.

2.10. Analytical.

Plasma and brain samples were prepared as described previously to analyze GAT229 and ABX-1431 concentrations (Anderson et al., 2020, 2019b). GAT229 samples were reconstituted in 0.1% formic acid in water and methanol (1:1, v/v) and ABX-1431 samples were reconstituted in 0.1% formic acid in water and acetonitrile (1:1, v/v) for analysis. Samples were assayed by LC-MS/MS with the following mass transition pairs: m/z 343.1 > 296.3, 343.1 > 282.1 and 343.1 > 193.2 (GAT229), 508.0 > 228.3 and 508.0 > 166.2 (ABX-1431). Quantification was achieved by comparing experimental samples to standards prepared with known amounts of compound.

3. RESULTS

3.1. Seizure-susceptible F1 (129 x B6) mice had lower hippocampal cannabinoid CB1 receptor expression than seizure-resistant (129) mice

Expression of the Scn1a+/− phenotype is strain-dependent with 129.Scn1a+/− mice having no overt phenotype and F1.Scn1a+/− mice exhibiting a severe epilepsy phenotype. We compared the expression of components within the endocannabinoid system across mouse background strains to assess whether the endocannabinoid system could be contributing to the strain-dependent effects. First, transcript expression of Cnr1, which encodes the CB1 receptor, was examined in the cortex and hippocampus (Figure 1A, B). There were no differences in cortical Cnr1 mRNA expression across groups; however, in the hippocampus a strain-dependent effect was observed with wildtype and Scn1a+/− mice on the seizure-susceptible F1 (129 x B6) background strain having significantly less Cnr1 mRNA than mice on the seizure-resistant 129 background strain (F1,31 = 7.178, p = 0.0117; two-way ANOVA). We next measured CB1 receptor protein expression in wildtype and Scn1a+/− mice on both background strains. Again, an overall strain-dependent effect was observed (F1,23 = 5.181, p = 0.0325; three-way ANOVA; Figure 1C). Because no effect of sex on CB1 receptor expression was observed, males and females were combined for further analysis (Figure 1D, E). The lower Cnr1 mRNA expression in F1 (129 x B6) mice correlated with a significantly lower CB1 receptor protein expression (F1,27 = 6.026, p = 0.0208; two-way ANOVA). Together, these results led to the consideration of Cnr1 as a candidate modifier gene.

Figure 1. Strain-specific cannabinoid receptor expression.

Figure 1.

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Relative Cnr1 transcript levels in the (A) cortex and (B) hippocampus of wildtype (WT, open bars) and Scn1a+/− (grey bars) mice on 129 and F1 (129 x B6) background strains. Cnr1 expression was measured in primary RNA pools using RT-ddPCR and expressed as a ratio of Tbp. Data represents mean ± SEM, with n = 7–11 per group. Hippocampal Cnr1 expression was significantly lower in F1 compared to 129 mice (*p < 0.05, two-way ANOVA). (C) Densitometry analysis of CB1 receptor expression as a ratio to β-tubulin for individual male (blue) and female (purple), wildtype (WT, open bars) and Scn1a+/− (shaded bars) mice on 129 and F1 (129 x B6) background strains. A significant strain-dependent effect was observed (p < 0.05, three-way ANOVA). Data represents mean ± SEM, with n = 3–4 per group. (D) Males and females in panel C were combined because no-sex dependent effect was observed. Hippocampal CB1 receptor expression was significantly lower in male and female F1 compared to 129 mice (*p < 0.05, two-way ANOVA). Data represents mean ± SEM, with n = 7–8 per group. (E) Western blot analysis of CB1 receptor levels in hippocampal membrane preparations from WT and Scn1a+/− (KO) mice on 129 and F1 (129 x B6) background strains. Representative blots from two biological replicates are shown for each background strain and genotype. Image was cropped to improve conciseness. Full-length Western blot image can be found in Supplemental Figure 1.

3.2. Brain endocannabinoids concentrations were equivalent in seizure-susceptible F1 (129 x B6) mice and seizure-resistant (129) mice

We compared hippocampal concentrations of the major endocannabinoids and components of their synthetic and degradative pathways (Figure 2A, Supplemental Figure 2). Hippocampal concentrations of 2-arachidonoylglycerol (2-AG), 1-arachidonoylglycerol (1-AG) and anandamide (AEA) were measured in wildtype and Scn1a+/− mice on both background strains in both sexes. No isomerization of 2-AG to 1-AG was observed with our endocannabinoid preparation method. Thus, the 1-AG concentrations measured here reflect 1-AG concentrations in the samples. Strain by sex-dependent effects were observed for both 2-AG and 1-AG (2-AG: F1,31= 6.914, p = 0.0130; 1-AG: F1,31 = 7.908, p = 0.0085; three-way ANOVA; Figure 2B, C). Since genotype had no effect on 2-AG or 1-AG concentrations, genotype data for each was combined. Strain by sex effects were again observed (2-AG: F1,35 = 7.3, p = 0.0104; 1-AG: F1,35 = 7.2, p = 0.0107; two-way ANOVA); however, no significant effects were apparent with Tukey’s post hoc analysis. Anandamide concentrations were not different across groups; however, there was substantial variability in the anandamide data (Figure 2D). Additionally, several of the data points were below the limit of detection (LOD) for anandamide, so a value of 0.0625 pmol/mg brain (one-half the LOD) was used for these samples.

Figure 2. Hippocampal concentrations of major endocannabinoids.

Figure 2.

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(A) Chemical structures of endocannabinoids. Hippocampal concentrations of (B) 2-arachidonoylglycerol, (C) 1-arachidonoylglycerol and (D) anandamide in male (blue) and female (purple), wildtype (WT, open bars) and Scn1a+/− (shaded bars) mice on 129 and F1 (129 x B6) background strains. A significant strain by sex interaction was observed for both 2-AG and 1-AG (left panels; p < 0.05; three-way ANOVA). Genotypes (WT and Scn1a+/−) were combined for respective groups (right panels). Significant interaction effects were present from both 2-AG and 1-AG (p < 0.05; two-way ANOVA). Statistical comparisons of 1-AG concentrations were made using 1/concentration values. No differences across groups were observed for anandamide concentrations. Dashed line represents the limit of detection for anandamide (LOD 0.125 pmol/mg brain), with data points on the line having anandamide concentrations below the limit of detection. Data represents mean ± SEM, with n = 4–5 per group.

3.3. Enhancing endocannabinoid tone was anticonvulsant against hyperthermia-induced seizures

We hypothesized that the reduced CB1 receptor expression in F1 (129 x B6) mice contributes to the strain-dependent phenotype severity observed in Scn1a+/− mice. To ascertain whether this difference within the endocannabinoid system is physiologically relevant to the phenotype of F1.Scn1a+/− mice we used two distinct pharmacological strategies to enhance endocannabinoid tone: 1) positive allosteric modulation of CB1 receptors, and 2) inhibition of monoacylglycerol lipase (MAGL), the primary enzyme responsible for the degradation of 2-AG.

The effect of GAT229, a CB1 receptor positive allosteric modulator (PAM), against hyperthermia-induced seizures in F1.Scn1a+/− mice was examined. We first conducted pharmacokinetic analysis to guide the assessment of its anticonvulsant potential. GAT229 was rapidly absorbed following an i.p. injection and achieved low micromolar brain concentrations (Figure 3A, B; Table 1). GAT229 was then evaluated for efficacy against hyperthermia-induced seizures and was anticonvulsant (Figure 3C). GAT229 at 30 and 100 mg/kg doses significantly elevated the body temperature threshold at which mice had a generalized tonic-clonic seizure (GTCS) compared to vehicle treatment (p = 0.0152 and p = 0.0059, respectively). Plasma concentrations of GAT229 following 30 mg/kg and 100 mg/kg treatments were not different, which is not surprising since both doses were administered as suspensions due to poor aqueous solubility (Table 2).

Figure 3. GAT229 and ABX-1431 are anticonvulsant against hyperthermia-induced seizures in F1.Scn1a+/− mice.

Figure 3.

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Concentration-time curve for GAT229 in mouse (A) plasma and (B) brain following a 5 mg/kg i.p. injection. Data are expressed as means ± SEM, with n = 4–5 per time point. (C) Threshold temperature of individual mice for GTCS induced by hyperthermia following acute i.p. treatment with vehicle (grey bar) or varying doses of GAT229 (green bars). GAT229 (30 mg/kg and 100 mg/kg) significantly elevated the temperature thresholds (41.7 ± 0.2°C and 41.8 ± 0.1°C, respectively) for thermally-induced seizures compared to vehicle (41.1 ± 0.1°C). The average temperatures of seizure induction are depicted by the bars and error bars represent SEM, with n = 18–22 per group (*p < 0.05, **p < 0.01; logrank Mantel-Cox). Concentration-time curve for ABX-1431 in mouse (D) plasma and (E) brain following a 5 mg/kg i.p. injection. Data are expressed as means ± SEM, with n = 4–5 per time point. (F) Threshold temperature of individual mice for GTCS induced by hyperthermia following acute i.p. treatment with vehicle (grey bar) or varying doses of ABX-1431 (blue bars). ABX-1431 (10 mg/kg) significantly elevated the temperature threshold (41.3 ± 0.2°C) for thermally-induced seizures compared to vehicle (40.6 ± 0.1°C). The average temperatures of seizure induction are depicted by the bars and error bars represent SEM, with n = 17 per group (***p < 0.001; logrank Mantel-Cox). Hippocampal concentrations of (G) 2-AG and (H) AEA in F1.Scn1a+/− mice from thermally induced seizure experiments. There were no difference in endocannabinoid concentrations between male and female vehicle-treated mice so sexes were combined for all groups. ABX-1431 treatment resulted in a dose-dependent increase in 2-AG concentrations (*p < 0.05, **p < 0.01, Brown-Forsythe ANOVA followed by Dunnett’s T3 post hoc). No differences were observed for anandamide concentrations with acute ABX-1431 treatment (Kruskal-Wallis test). Data represents mean ± SEM, with n = 5–10 per group. (I) Threshold temperature of individual mice for GTCS induced by hyperthermia following acute treatment with ABX-1431 (blue bar) and GAT229 (green bar) administered individually or in combination (blue-grey bar). ABX-1431 (10 mg/kg) and GAT229 (30 mg/kg) significantly elevated the temperature threshold for thermally-induced seizures. The average temperatures of seizure induction are depicted by the bars and error bars represent SEM, with n = 16 per group (*p < 0.05; logrank Mantel-Cox).

Table 1.

Pharmacokinetic parameters in mouse plasma and brain

ABX-1431 GAT229
Plasma Brain Plasma Brain
Cmax (ng/mL) 468 ± 40 317 ± 40 858± 167 888± 90*
tmax (ng/mL) 30 120 30 30
t1/2 (min) 47 not determined 45 53
AUC (μg min/mL) 29 not determined 74 85*
brain-plasma not determined 110%
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*

concentrations (ng/mL) converted from measured concentrations (ng/mg brain) assuming density of 1 g/mL

Table 2.

Compound concentrations in experimental mice

Plasma Concentration (ng/mL)
ABX-1431
3 mg/kg 198 ± 31
10 mg/kg 373 ± 53
30 mg/kg 883 ± 114
350 mg/kg chow 170 – 394
GAT229
10 mg/kg 0.9 ± 0.1 μg/mL
30 mg/kg 2.6 ± 0.5 μg/mL
100 mg/kg 3.2 ± 0.2 μg/mL
GAT229 + ABX-1431 (30 mg/kg + 10 mg/kg)
GAT229: 2.3 ± 0.3 μg/mL
ABX-1431: 369 ± 80
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Next, we examined the effect of ABX-1431, a MAGL inhibitor, against hyperthermia-induced seizures in F1.Scn1a+/− mice. A pharmacokinetic analysis of ABX-1431 was conducted first (Figure 3D, E; Table 1). ABX-1431 was readily absorbed and attained high nanomolar brain concentrations. Notably, ABX-1431 had a prolonged brain half-life, maintaining high concentrations until the final 180 min sampling time. ABX-1431 was also anticonvulsant against hyperthermia-induced seizures in F1.Scn1a+/− mice (Figure 3F). However, ABX-1431 treatment exhibited an inverse U-shaped effect, where 10 mg/kg significantly elevated the GTCS temperature threshold compared to vehicle (p = 0.0008) whilst 30 mg/kg did not. Importantly, ABX-1431 treatment dose-dependently increased hippocampal 2-AG concentrations (F*3,12.69= 10.66, p < 0.001; Brown-Forsythe ANOVA; Figure 3G). The effect of ABX-1431 appeared to be specific to 2-AG, as anandamide concentrations were not affected (Figure 3H).

Next, F1.Scn1a+/− mice were co-treated with GAT229 and ABX-1431 to determine whether the anticonvulsant effects could be enhanced. Replicating our prior results, treatment with 10 mg/kg ABX-1431 and 30 mg/kg GAT229 significantly elevated the GTCS temperature threshold compared to vehicle treatment (ABX-1431: p = 0.0123; GAT229: p = 0. 0263; Figure 3I). Combination treatment with 10 mg/kg ABX-1431 and 30 mg/kg GAT229, however, did not affect the temperature threshold for hyperthermia-induced seizures (p = 0.1449).

3.4. Subchronic oral treatment with the MAGL inhibitor ABX-1431 increases spontaneous seizure frequency

Dravet syndrome typically presents with febrile seizures that progress to other seizure types including spontaneous GTCS, which are replicated in F1.Scn1a+/− mice (Hawkins et al., 2017). Additionally, Dravet syndrome patients and F1.Scn1a+/− mice have a reduced lifespan. We aimed to investigate the effect of GAT229 and ABX-1431 on spontaneous seizure frequency and survival of F1.Scn1a+/− mice. In this paradigm, treatments are administered subchronically via supplementation of compound into chow to avoid the stress associated with repeated i.p. injections. Unfortunately, GAT229 appears to exhibit poor oral bioavailability, as sufficient plasma GAT229 concentrations were not achieved following subchronic oral administration. As such, spontaneous seizure and survival experiments with GAT229 were not conducted.

The subchronic oral ABX-1431 formulation (350 mg ABX-1431/kg chow) resulted in steady-state plasma ABX-1431 concentrations equivalent to those achieved following acute i.p. treatment with 10 mg/kg ABX-1431 (Table 2). ABX-1431 treatment did not affect the proportion of F1.Scn1a+/− mice that exhibited seizures (p = 0.6562); however, it did significantly increase spontaneous seizure frequency compared to untreated mice (p = 0.0064; Figure 4A). Interestingly, despite ABX-1431 treatment increasing GTCS frequency, it also reduced the severity of the seizures compared to untreated mice (p = 0.0011; Figure 4B), as measured by progression to the most severe stage of full hindlimb extension. The proportion of seizures that progressed to full hindlimb extension was 45/83 (54%) in untreated mice, whereas in ABX-1431-treated mice the proportion was only 93/272 (34%). Despite subchronic ABX-1431 treatment resulting in increased GTCS frequency, survival of F1.Scn1a+/− mice was not different from untreated controls (p = 0.6803), likely a result of the reduced seizure severity (Figure 4C). As expected, hippocampal 2-AG concentrations were significantly higher following subchronic ABX-1431 treatment (p = 0.0314, Figure 4D). Interestingly, subchronic ABX-1431 treatment significantly lowered hippocampal CB1 receptor expression (p = 0.0077, Figure 4E).

Figure 4. ABX-1431 is proconvulsant against spontaneous seizures of F1.Scn1a+/− mice.

Figure 4.

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(A) Generalized tonic-clonic seizure (GTCS) frequency of individual untreated and ABX-1431-treated mice. Treatment was administered orally via supplementation in chow at P18 following the induction of a single hyperthermia-induced seizure. Unprovoked, spontaneous GTCS were quantified over a 60-h recording period. ABX-1431 treatment significantly increased the frequency of GTCS, with n = 17 per group (**p < 0.01, Mann-Whitney test). (B) Proportion of spontaneous GTCS with (grey bars) or without (white bars) full tonic hindlimb extension. ABX-1431 treatment significantly reduced the severity of GTCS (**p < 0.005, Fisher’s exact test). Total number of seizures for each treatment is presented in parentheses above the bar. (C) Survival curves comparing untreated and ABX-1431-treated mice. Treatment began at P18 and survival was monitored to P30. ABX-1431 had no effect on survival of F1.Scn1a+/− mice (Mantel-Cox logrank). (D) Hippocampal concentrations of 2-AG in F1.Scn1a+/− mice untreated or treated with ABX-1431. Concentrations of 2-AG are significantly increased following subchronic ABX-1431 treatment (*p < 0.05, Welch’s t-test). Data represents mean ± SEM, with n = 4 per group. (E) Densitometry analysis of CB1 receptor expression as a ratio to β-tubulin for individual F1.Scn1a+/− mice untreated or treated with ABX-1431. CB1 receptor expression was significantly reduced following subchronic ABX-1431 treatment (**p < 0.01, Student’s t-test). Data represents mean ± SEM, with n = 4 per group. Full-length Western blot image can be found in Supplemental Figure 3.

4. DISCUSSION

There is an urgent need to develop improved therapeutic options for treatment resistant epilepsies, including Dravet syndrome. The Scn1a+/− mouse model of Dravet syndrome has been established as a translationally validated platform to screen potential Dravet syndrome treatments. Not only do Scn1a+/− mice exhibit the hallmark features of Dravet syndrome, but their response to currently available anticonvulsants is consistent with those of Dravet syndrome patients (Anderson et al., 2019a; Hawkins et al., 2017; Kaplan et al., 2017; Patra et al., 2019). In addition to being an effective screening platform, the Scn1a+/− mouse model can also be used to discover potential novel drug targets by identifying genetic modifiers The present study used the Scn1a+/− mouse model of Dravet syndrome to compare the expression of components in the endocannabinoid system between seizure-susceptible and seizure-resistant genetic background strains, which identified Cnr1 as a putative genetic modifier of Scn1a+/− mice. Subsequently, we used two distinct pharmacological strategies to assess the anticonvulsant potential of augmenting endocannabinoid system tone in F1.Scn1a+/− mice: positive allosteric modulation of CB1 receptors and MAGL inhibition to increase 2-AG concentrations. Both approaches were anticonvulsant against hyperthermia-induced seizures in F1.Scn1a+/− mice. However, despite reducing spontaneous seizure severity, prolonged and greatly augmented 2-AG concentrations increased seizure frequency, highlighting a potential liability of enhanced endocannabinoid tone in treating Dravet syndrome.

We showed that the CB1 receptor PAM GAT229 had anticonvulsant effects in a mouse model of intractable childhood epilepsy, where it significantly elevated the body temperature threshold for hyperthermia-induced seizures in F1.Scn1a+/− mice at both 30 and 100 mg/kg. Plasma concentrations of GAT229 in these experimental animals were 7.6–9.3 μM (2.6–3.2 μg/mL). Our pharmacokinetic study showed that GAT229 exhibited great brain penetrance with a brain-plasma ratio of 1.1. Based on this value of 110% brain penetration, the estimated brain concentrations of GAT229 following 30 and 100 mg/kg doses would be 9.4 to 10.2 μM. Previous work has shown that GAT229 has an EC50 value of approximately 295 nM at the CB1 receptor (Laprairie et al., 2017). Thus, the concentrations of GAT229 achieved with these anticonvulsant doses would be well within its therapeutic range at CB1 receptors even if GAT229 were to exhibit high protein binding (i.e. 3% unbound). Notably, GAT229 was a more potent anticonvulsant than CBD which is effective at 100 mg/kg in this model (Anderson et al., 2020, 2019a; Kaplan et al., 2017). GAT229 was inferior, however, to the first-line treatment clobazam, which is effective at 1 mg/kg (Anderson et al., 2019a; Hawkins et al., 2017). Recently, GAT229 also demonstrated anticonvulsant effects against absence seizures in Genetic Absence Epilepsy Rats from Strasbourg (GAERS)(Roebuck et al., 2021). Thus, it appears that CB1 receptor positive allosteric modulation may be a viable and novel means of achieving anticonvulsant effects across different epilepsy types. Future studies could continue to assess the effects of GAT229 and new generation CB1 receptor PAMs in additional epilepsy models to extend the evaluation of this class of compounds.

CB1 receptor PAMs may offer a means of delivering anticonvulsant effects, without the intoxicating effects observed with orthosteric agonists such as Δ9-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis. CB1 receptor PAMs have no intrinsic activity at CB1 receptors as they do not bind the orthosteric site, but rather selectively potentiate endocannabinoids tone through allosteric modulation (Laprairie et al., 2017). In the GAERs model, GAT229 was not associated with sedation or catalepsy (Roebuck et al., 2021). However, a previous study showed reduced locomotor activity of wildtype mice treated with 10 mg/kg GAT229 (Laprairie et al., 2019).

The anticonvulsant efficacy of GAT229 did not show clear dose-dependency. The plateau in efficacy could simply be the result of limitations in the solubility of GAT229 with doses of 30 mg/kg and 100 mg/kg administered as suspensions and yielding roughly equivalent plasma and brain GAT229 concentrations. Alternatively, the plateau could be the result of a “ceiling effect”, often observed with allosteric modulators, where a functional maximal asymptote of positive allosteric modulation is reached upon complete occupancy of the allosteric site at a given orthosteric agonist concentration.(Kenakin, 2017) Here we attempted to surmount a possible “ceiling effect” of GAT229 by increasing concentrations of the endogenous orthosteric agonist 2-AG. This strategy promoted a synergistic antinociceptive effect when GAT211, the racemic mixture of GAT229, was combined with the MAGL inhibitor JZL184 in a neuropathic pain model (Slivicki et al., 2018). However, combination treatment with active doses of GAT229 and ABX-1431 did not potentiate the anticonvulsant effects of either compound; in fact, it appeared that there was interference between GAT229 and ABX-1431 as combination treatment was not anticonvulsant.

Unfortunately, we were unable to examine the anticonvulsant potential of GAT229 against spontaneous GTCS and survival in F1.Scn1a+/− mice because sufficient steady-state plasma concentrations could not be achieved following subchronic oral administration. GAT229 has poor aqueous solubility, which predicts low oral bioavailability (Martinez and Amidon, 2002). These limitations would need to be overcome to progress the development of GAT229 as an anticonvulsant. Next-generation CB1 receptor PAMs are being developed to improve physicochemical and pharmacokinetic properties (Garai et al., 2020; Tseng et al., 2019). However, the next-generation compounds have been limited to those with combined PAM activity and CB1 receptor agonism at higher concentrations (so called ago-PAMs). While intoxicating effects would be expected with a CB1 receptor ago-PAM, canonical cannabimimetic effects were not observed at therapeutically relevant doses in mouse models of analgesia (Badowski, 2017; Ignatowska-Jankowska et al., 2015; Sam et al., 2011; Slivicki et al., 2018). Future studies may explore these new CB1 receptor ago-PAMs for anticonvulsant potential in the F1.Scn1a+/− mouse model of Dravet syndrome.

MAGL inhibitors display anticonvulsant effects in conventional seizure models (Sugiura et al., 2006). Here, we demonstrate the highly selective, irreversible MAGL inhibitor ABX-1431 displayed mixed anticonvulsant and proconvulsant activity in a mouse model of intractable childhood epilepsy. ABX-1431 was anticonvulsant against hyperthermia-induced seizures in F1.Scn1a+/− mice, but it displayed an inverted U-shaped dose-effect, with an increase in GTCS temperature threshold at 10 mg/kg but not at 30 mg/kg. Subchronic treatment of F1.Scn1a+/− mice with ABX-1431 increased spontaneous GTCS frequency while decreasing seizure severity. The contrasting anticonvulsant and proconvulsant effects of MAGL inhibition presented here might reflect the presence of a therapeutic window for 2-AG elevation. At the anticonvulsant 10 mg/kg i.p. dose, we observed hippocampal 2-AG concentrations 8-fold higher than vehicle-treated mice. Whereas, 2-AG concentrations increased 13 and 15-fold with following acute 30 mg/kg i.p and the subchronic oral (350 mg/kg chow) ABX-1431 treatments, respectively. Thus, it is possible that an optimal increase in 2-AG concentrations is required for anticonvulsant efficacy and that excessive 2-AG concentrations become ineffective or proconvulsant.

The endocannabinoid system contributes to both excitatory and inhibitory neurotransmission. Concentration-dependent effects of 2-AG on glutamatergic and GABAergic signaling might, thus, explain the contrasting anticonvulsant and proconvulsant effects of ABX-1431 observed here. That is, a relatively small increase in 2-AG concentrations might be anticonvulsant by selectively dampening excitatory glutamate neurotransmission; whereas, larger increases in 2-AG concentrations might additionally activate CB1 receptors on inhibitory GABAergic interneurons thereby increasing neuronal hyperexcitability via disinhibition. Indeed, this has been observed for other MAGL inhibitors, where low doses promote CB1 receptor-dependent long-term depression at glutamatergic synapses, and high doses more prominently elicited CB1 receptor-dependent inhibition of GABAergic interneurons.(Wang et al., 2017) Therefore, subchronic ABX-1431 treatment may have preferentially activated CB1 receptors on inhibitory GABAergic interneurons resulting in neuronal hyperexcitability.

Additionally, it should be considered that thermally-induced seizures upregulate CB1 receptor expression on GABAergic interneurons, and that F1.Scn1a+/− mice were primed with a hyperthermia-induced seizure before quantifying spontaneous GTCS (Chen et al., 2003). Further, higher 2-AG concentrations are known to activate excitatory receptors such as adenosine A3 receptors, GPR55, TRPA1 and TRPV1 receptors, providing an additional mechanism for ABX-1431-induced seizures (Günaydın et al., 2020; Heblinski et al., 2020; Long et al., 2009a; Petrosino et al., 2016; Ryberg et al., 2007).

The proconvulsant effects of ABX-1431 on spontaneous seizures might also be explained by CB1 receptor desensitization and downregulation as a result of prolonged brain exposure to supraphysiological 2-AG concentrations achieved with subchronic ABX-1431 treatment. Indeed, hippocampal CB1 receptor expression was lower following subchronic ABX-1431 treatment. CB1 receptor downregulation and desensitization has been reported in response to extended MAGL inhibition, which could diminish the negative feedback of 2-AG on glutamate release and result in increased network excitability (Chanda et al., 2010; Schlosburg et al., 2010). Recently, two reversible MAGL inhibitors were developed, which elicit more transient increases in 2-AG concentrations than irreversible inhibitors like ABX-1431 and have a purported reduced capacity to induce CB1 receptor downregulation and desensitization (Aida et al., 2018; Granchi et al., 2019). However, CB1 receptor downregulation and desensitization does not occur with repeated exposure to low doses of the irreversible MAGL inhibitor JZL184 (Ghosh et al., 2013; Kinsey et al., 2013). Spontaneous seizure experiments could be repeated using a lower dose of ABX-1431 or a reversible MAGL inhibitor to further explore the anticonvulsant potential of subchronic MAGL inhibition in F1.Scn1a+/− mice.

The endocannabinoid system has not been extensively characterized in Dravet syndrome patients. One study has reported an increased expression of cannabinoid CB2 receptors in lymphocytes of patients and no change in plasma endocannabinoid concentrations (Rubio et al., 2016). Cell-specific alterations in brain endocannabinoid signalling has only been characterized in refractory temporal lobe epilepsy patients (TLE), where TLE patients had significantly reduced hippocampal expression of the CB1 receptor and DAGLα, the predominant 2-AG biosynthetic enzyme, on glutamatergic axon terminals and elevated CB1 receptor expression on inhibitory axon terminals (Goffin et al., 2011; Ludanyi et al., 2008). Thus, concurrent upregulation of CB1 receptors on GABAergic terminals and downregulation of CB1 receptors on glutamatergic terminals may contribute to the mechanism of seizures in humans. Future studies might examine CB1 receptor expression on glutamatergic versus GABAergic terminals in Scn1a+/− mice and post-mortem brain of Dravet syndrome patients. Research may also attempt to confirm whether CNR1 is a modifier in Dravet syndrome patients.

In summary, we identified Cnr1 as a putative genetic modifier in the Scn1a+/− mouse model of Dravet syndrome. Positive allosteric modulation of the CB1 receptor and enhancing brain 2-AG concentrations via MAGL inhibition both yielded anticonvulsant effects against hyperthermia-induced seizures in Scn1a+/− mice, suggesting these endocannabinoid system targeting strategies may be viable for the development of next-generation anticonvulsant drugs. However, MAGL inhibition as an anticonvulsant mechanism may be limited by having a narrow therapeutic range, since we found that subchronic treatment with a high dose of ABX-1431 worsened spontaneous seizures. These data suggest that compounds that enhance endocannabinoid tone via the CB1 receptor could be developed as novel treatments for Dravet syndrome, a refractory childhood epilepsy.

Supplementary Material

Supplementary Material

ACKNOWLEDGEMENTS

This research was supported by the Lambert Initiative for Cannabinoid Therapeutics, a philanthropically-funded centre for medicinal cannabis research at the University of Sydney, the Australian National Health and Medical Research Council (GNT1161571) and the U.S National Institutes of Health (R01 NS084959). The authors gratefully acknowledge Barry and Joy Lambert for their continued support of the Lambert Initiative for Cannabinoid Therapeutics.

Footnotes

CONFLICTS OF INTEREST

Associate Professor Arnold has served as an expert witness in various medicolegal cases involving cannabis and cannabinoids and serves as a temporary advisor to the World Health Organization (WHO) on their review of cannabis. Associate Professor Arnold and Dr Anderson hold patents on cannabinoid therapies (PCT/AU2018/05089 and PCT/AU2019/050554). Dr Thakur holds a patent on allosteric modulators of CB1 cannabinoid receptors (US 9,556,118). The remaining authors have no conflicts of interest.

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