ORIGINAL ARTICLE Analysis of endocannabinoid signaling elements and related proteins in lymphocytes of patients with Dravet syndrome Marta Rubio1,2,3,a, Sara Valdeolivas1,2,3,a, Fabiana Piscitelli4, Roberta Verde4, Valentina Satta1,2,3, Eva Barroso5,6, Marisol Montolio7,8, Luis Miguel Aras7,9, Vincenzo Di Marzo4, Onintza Sagredo1,2,3,b ndez-Ruiz1,2,3,b & Javier Ferna 1 n en Neuroquımica, Universidad Departamento de Bioquımica y Biologıa Molecular, Facultad de Medicina, Instituto Universitario de Investigacio Complutense, Madrid, Spain 2 Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 3 Instituto Ramon y Cajal de Investigacio n Sanitaria (IRYCIS), Madrid, Spain 4 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy 5 Instituto de Genetica Medica y Molecular (INGEMM), Hospital Universitario La Paz, Universidad Autonoma de Madrid, IdiPAZ, Madrid, Spain 6 Centro de Investigacion Biomedica en Red sobre Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain 7 Dravet Syndrome Foundation, Madrid, Spain 8 Departamento de Biologıa Celular, Facultad de Biologıa, Universidad de Barcelona, Barcelona, Spain 9 Servicio Navarro de Salud, Osasunbidea, Estella, Spain Keywords Abstract Dravet syndrome, endocannabinoid signaling, endocannabinoids, lymphocytes Cannabidiol (CBD) reduces seizures in childhood epilepsy syndromes including Dravet syndrome (DS). A formulation of CBD has obtained orphan drug desig- Correspondence nation for these syndromes and clinical trials are currently underway. The Javier Fern andez-Ruiz or Onintza Sagredo, mechanism responsible for CBD effects is not known, although it could involve Departamento de Bioquımica y Biologıa targets sensitive to CBD in other neurological disorders. We believe of interest Molecular, Facultad de Medicina, Universidad to investigate whether these potential targets are altered in DS, in particular Complutense, Madrid 28040, Spain. Tel: 34913941450 or 34913941454; whether the endocannabinoid system is dysregulated. To this end, lymphocytes Fax: 34913941691; E-mail: jjfr@med.ucm.es from patients and controls were used for analysis of gene expression of trans- or onintza@med.ucm.es mitter receptors and transporters, ion channels, and enzymes associated with CBD effects, as well as endocannabinoid genes. Plasma endocannabinoid levels Funding Information were also analyzed. There were no differences between DS patients and controls This work was supported by grants from in most of the CBD targets analyzed, except an increase in the voltage-depen- CIBERNED CB06/05/0089, MINECO dent calcium channel a-1h subunit. We also found that cannabinoid type-2 (SAF2012/39173) and CAM (S2011/BMD- (CB2) receptor gene expression was elevated in DS patients, with no changes in 2308) other endocannabinoid-related receptors and enzymes, as well as in plasma Received: 9 December 2015; Accepted: 20 levels of endocannabinoids. Such elevation was paralleled by an increase in January 2016 CD70, a marker of lymphocyte activation, and certain trends in inflammation- related proteins (e.g., peroxisome proliferator-activated receptor-c receptors, Pharma Res Per, 4(2), 2016, e00220, cytokines). In conclusion, together with changes in the voltage-dependent cal- doi: 10.1002/prp2.220 cium channel a-1h subunit, we found an upregulation of CB2 receptors, associ- ated with an activation of lymphocytes and changes in inflammation-related doi: 10.1002/prp2.220 genes, in DS patients. Such changes were also reported in inflammatory disor- a Both authors share the first authorship. ders and may indirectly support the occurrence of a potential dysregulation of the endocannabinoid system in the brain. b Both authors share the senior authorship and conducted the statistical assessment of Abbreviations the data. 2-AG, arachidonoylglycerol; 5HT1A, 5-hydroxytriptamine 1A receptor; 5HTT, sero- tonin transporter; A2A, adenosine 2A receptor; CBD, cannabidiol; CNS, central nervous system; COX-2, cyclooxygenase-2; DAGL, diacylglycerol lipase; DAT, ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2016 | Vol. 4 | Iss. 2 | e00220 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Peripheral Endocannabinoid Signaling and Dravet Syndrome M. Rubio et al. dopamine transporter; DS, Dravet syndrome; GABAT, GABA transporter; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter type-1; GPR18, G protein-coupled receptor 18; GPR55, G protein-coupled receptor 55; GUSB, glu- curonidase-B; IL-1, interleukin-1; Keap-1, Kelch-like ECH-associated protein 1; LC- APCI, liquid chromatography/atmospheric pressure chemical ionization; MAGL, monoacylglycerol lipase; NAPE-PLD, N-arachidonoyl-phosphatidylethanolamine- phospholipase D; NFjB, nuclear factor of j-light polypeptide gene enhancer in B-cells; NOS, nitric oxide synthase; Nrf-2, nuclear factor, erythroid 2-like 2; OEA, oleylethanolamide; PEA, palmitoylethanolamide; PPAR, peroxisome proliferator- activated receptor; TNF, tumor necrosis factor; TRPA1, transient receptor potential ankyrin type-1; TRPV1, transient receptor potential vanilloid type-1; TRPV2, tran- sient receptor potential vanilloid type-2. Jacobson 2013; Devinsky et al. 2014a), which targets indi- Introduction rectly the endocannabinoid system and directly proteins Dravet syndrome (DS), also known as severe myoclonic outside this signaling system (Fernandez-Ruiz et al. 2013). epilepsy of infancy, was described in 1978 by Charlotte CBD had been already investigated for its anticonvulsant Dravet (Dravet 1978). DS is a rare genetic pediatric epi- properties in seizure models of adult epilepsy (Hill et al. lepsy whose incidence is estimated in 1:20,000 subjects and 2012), but the first evidence of its potential for infantile it occurs more often in males than in females (2:1) (Hurst epileptic syndromes was collected in 2013, when a num- 1990). DS typically presents around 5–8 months of age ber of US families having children affected by DS or with febrile seizures that progress to severe partial or gen- related epileptic syndromes used CBD-enriched cannabis eralized tonic–clonic seizures, myoclonic seizures, and epi- to attenuate the frequency and intensity of epileptic sodes of status epilepticus. It is often accompanied by episodes. A follow-up survey collected their experience cognitive impairment, behavioral disturbances with hyper- indicating a >80% reduction in seizure frequency in activity, and sometimes autistic traits (Guerrini 2012). In approximately 42% of children treated with CBD- approximately 60–80% of patients, DS is caused by a enriched cannabis (Porter and Jacobson 2013), although mutation in SCN1A, the gene encoding the a-subunit of the preparation also included certain quantities of D9-tet- the voltage-gated sodium channel NaV1.1 whose function rahydrocannabinol which has dual effects on epileptic sei- contributes to the rising phase of the action potential zures (Hill et al. 2012). Despite the anecdotal nature of (Bender et al. 2012). The use of mouse models of DS has these noncontrolled data, and that the issue had not been improved our understanding of those mechanisms by investigated in DS mouse models yet, an oral formulation which mutations in SCN1A gene may lead to seizure activ- of CBD developed by the British company GW Pharma- ity and cognitive dysfunction (Cheah et al. 2012). Loss of ceuticals received Orphan Drug Designations by the US function in Nav1.1 causes impaired firing of GABAergic Food and Drug Agency and by the European Medical interneurons which results in an imbalance between excita- Agency for the treatment of childhood epilepsy syn- tion and inhibition, leading to seizures (Cheah et al. dromes. Then, clinical trials were initiated in the US and 2012), although a direct alteration in excitatory pyramidal Europe (www.gwpharma.com/Epidiolex.aspx), and pre- neurons has been also proposed (Mistry et al. 2014). In liminary data indicate that a relevant number of patients both cases, these functional deficits likely alter the normal achieved seizure disappearance or experienced a notable function of these neural networks known to be critical to reduction in their seizure frequency after the treatment cognitive functions (Bender et al. 2012). Despite these with CBD, as has been recently disclosed in the American advances, DS pathogenesis remains poorly understood and Epilepsy Society 2014 meeting (Devinsky et al. 2014b). only one drug, stiripentol, has been approved specifically The most intriguing aspect of these clinical studies is that for the treatment of this syndrome, although not in USA. they were initiated with no previous preclinical or clinical In general, patients are treated with combinations of classic evidence of the potential of CBD for DS and related syn- antiepileptic drugs (Kassa€ı et al. 2008) and ketogenic diet dromes, other than its well-known anticonvulsant proper- (Laux and Blackford 2013), but they remain largely phar- ties studied in preclinical models of adult epilepsy (Hill macoresistant. Therefore, novel therapies are urgently et al. 2012) and also in an old clinical trial that included needed for this epileptic syndrome. treatment-resistant patients (Cunha et al. 1980). In this Preliminary data suggest that a promising therapy for context, our present study attempted as a first objective to DS may be the use of cannabidiol (CBD) (Porter and investigate the changes occurring in DS in different 2016 | Vol. 4 | Iss. 2 | e00220 ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 2 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. M. Rubio et al. Peripheral Endocannabinoid Signaling and Dravet Syndrome pharmacological targets that have been directly or indi- were also used to analyze gene expression for different rectly related to numerous therapeutic effects of CBD seen endocannabinoid-related elements including receptors in other neurological disorders (Fernandez-Ruiz et al. (e.g., CB1 and CB2 receptors, G protein-coupled receptor 2013), so that we can collect any evidence that relates 55 [GPR55], G protein-coupled receptor 18 [GPR18], changes in a specific target to the expected benefits of CBD TRPV1, transient receptor potential vanilloid type-2 in DS. These targets included certain transmitter receptors [TRPV2], transient receptor potential ankyrin type-1 and transporters, as well as different enzymes and ion chan- [TRPA1]) and enzymes (e.g., N-arachidonoyl-phosphati- nels, which are not related to the endocannabinoid system dylethanolamine-phospholipase D [NAPE-PLD], fatty but have the additional interest of having been related to acid amide hydrolase [FAAH], diacylglycerol lipase epileptic states too (Hill et al. 2012; Fernandez-Ruiz et al. [DAGL], and monoacylglycerol lipase [MAGL]), as well 2013). This is in relation to recent evidence showing that as for some proteins downstream the endocannabinoid CBD may be active at micromolar concentrations on NaV signaling (e.g., NFjB, Akt, nuclear factor, erythroid 2-like (Hill et al. 2014; Patel et al. 2014) and transient receptor 2 [Nrf-2], Keap-1, b-arrestin-1, b-arrestin-2) and also potential vanilloid type-1 (TRPV1) (Iannotti et al. 2014) related to inflammation (e.g., peroxisome proliferator- channels, although it is not clear whether this activity activated receptor-c [PPAR-c], tumor necrosis factor-a underlies the anticonvulsant properties of CBD. We also [TNF-a], interleukin-1b [IL-1b], inducible NOS, included genes encoding for a few elements within the cyclooxygenase-2 [COX-2], CD70). In addition, we have endocannabinoid signaling system, despite CBD being an analyzed the levels of endocannabinoids and related lipid atypical cannabinoid with poor activity in relation to classic derivatives in the plasma of DS patients. We assume that cannabinoid type-1 (CB1) and type-2 (CB2) receptors changes in gene expression do not necessarily reflect sub- (Fernandez-Ruiz et al. 2013). Nevertheless, we wanted, as a sequent changes in protein levels, and this represents a second objective, to investigate how DS affects the endo- potential limitation of our study. However, the isolation cannabinoid signaling system. This may be an important goal of RNA samples for qRT-PCR analysis was the best and given the increasing evidence supporting a role of this signal- only way to analyze the maximal number of potential dis- ing system in epilepsy (Hill et al. 2012), as well as in other ease-related markers in blood lymphocytes, whose collec- neurological disorders in which a dysregulated endocannabi- tion is necessarily limited in children due to reasons of noid signaling has been related to specific events within dis- availability and ethical concerns. ease pathogenesis and/or to an adaptive response derived from disease progression (Massa et al. 2010; Alvaro-  Materials and Methods Bartolome and Garcıa-Sevilla 2013), in both cases having an influence on potential cannabinoid-based therapies. Patient recruitment Our study was carried out in control and DS patient lymphocytes. These immune cells are frequently used in All studies were conducted in peripheral blood mononu- different neurological disorders to recapitulate changes in clear cells (approximately 85% are lymphocytes), and specific markers that may also occur in the central ner- plasma samples obtained from DS patients (n = 15; vous system (CNS) (Arosio et al. 2014). However, the recruited by the Dravet Syndrome Foundation, Spain). changes found in these cells may be also interpreted as a The relatively low number of patients accounts for the response derived from the influence of the disease on the low incidence of DS, which is a rare disease Moreover, own immune system, in particular, from a repetitive sei- the disease affects infantile ages, which makes more com- zure activity that is expected, for example, to generate plicated the recruitment due to logistic and ethical rea- inflammatory responses. Lymphocytes were used to ana- sons. The numbers were also limited because we tried to lyze gene expression for: (i) ion channels, for example, get a certain homogeneity in the patient cohort, for voltage-dependent calcium channel a-1h subunit (CAC- example, with all individuals bearing confirmed mutations NA1h); (ii) transmitter receptors, for example serotonin- in SCN1A gene. They were compared with control sub- 1A receptor (5HT1A), adenosine 2A receptor (A2A); and jects (n = 10), who were non-DS individuals including (iii) transporters for glutamate (e.g., glutamate trans- some nonaffected relatives who do not bear the mutation porter type-1 [GLT-1], glutamate–aspartate transporter and have not undergone clinical diagnosis of DS. They [GLAST]), GABA (e.g., GABA transporter [GABAT]), were matched by age and gender to DS patients. The dopamine (e.g., Dopamine transporter [DAT]), serotonin study was approved by the “Comite Etico de Investi- (e.g., 5HTT), and adenosine (e.g., equilibrative nucleoside gacion Clınica,” IRYCIS, Madrid, Spain (code 389-13), transporter). In most of the cases, these proteins were and, in all cases, parents/legal representatives approved selected because they have been shown to be related to and signed an informed consent that discloses all ethical CBD action (Fernandez-Ruiz et al. 2013). Lymphocytes aspects related to this study. The major characteristics ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2016 | Vol. 4 | Iss. 2 | e00220 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 3 Peripheral Endocannabinoid Signaling and Dravet Syndrome M. Rubio et al. (age at sampling, gender, age at diagnosis, and some clini- those genes of interest (see details in Table 2; the nomen- cal information) of patients and control subjects are clature of all investigated targets including receptors, ion shown in Table 1. channels, transporters, etc., conforms to BJP’s Guide to Receptors and Channels [Alexander et al. 2011]) using glucuronidase-B (GUSB) expression as an endogenous Biological sampling control gene for normalization (the expression of this Blood samples from DS patients or control subjects were gene was similar in DS cases and controls and it was collected in BD Vacutainer Cell Preparation Tubes con- maintained relatively constant within the two groups: CT taining sodium citrate (BD, Franklin Lakes, NJ). Tubes values in controls: 27.30  0.46 [10]; in DS: 27.29  0.33 were inverted 8–10 times and centrifuged (1800 g) at [15]). The PCR assay was performed using the 7300 Fast room temperature for 25 min. Times between blood col- Real-Time PCR System (Applied Biosystems) and the lection and centrifugation were always shorter than threshold cycle (Ct) was calculated by the instrument’s 30 min. After centrifugation, the plasma was collected software (7300 Fast System, Applied Biosystems). Values and stored at 80°C for analysis of endocannabinoids were expressed as fold change over the control group and and related N-acylethanolamines. The layer containing were presented as mean  SEM. the lymphocytes was also collected and centrifuged again (300 g) at room temperature for 10 min. After this sec- Analysis of endocannabinoid levels ond centrifugation, the supernatant was removed and dis- carded, and the cell pellet stored at 80°C for analysis of Plasma samples were dounce-homogenized and extracted gene expression. in 5 vol of chloroform/methanol/Tris–HCl 50 mmol/L (2:1:1) containing 5 pmol of [2H]8 anandamide and 10 pmol of [2H]4 palmitoylethanolamide (PEA), [2H]2 oley- Real-time qRT-PCR analysis lethanolamide (OEA) and [2H]5 2-arachidonoylglycerol Total RNA was extracted from isolated lymphocytes using (2-AG) (Cayman Chemicals, Ann Arbor, MI). The lipid- SurePrepTM RNA/Protein Purification kit (Fisher BioRea- containing organic phase was dried down in a rotating gents, Fair Lawn, NJ). The total amount of RNA extracted evaporator, weighed, and prepurified by open-bed chro- was quantitated by spectrometry at 260 nm and its purity matography on silica gel. Lyophilized extracts were resus- was evaluated by the ratio between the absorbance values pended in chloroform/methanol 99:1 by vol. The at 260 and 280 nm, whereas its integrity was confirmed solutions were then purified by open-bed chromatography in agarose gels. To prevent genomic DNA contamination, on silica as described (Bisogno et al. 1997). Fractions DNA was removed and single-stranded complementary eluted with chloroform/methanol 9:1 by vol. (containing DNA was synthesized from 0.2 lg of total RNA using a anandamide, 2-AG, OEA, and PEA) were collected and commercial kit (Rneasy Mini Quantitect Reverse Tran- the excess solvent evaporated with a rotating evaporator, scription, Qiagen, Izasa, Madrid, Spain). The reaction and aliquots analyzed by isotope dilution-liquid chro- mixture was kept frozen at 20°C until enzymatic ampli- matography/atmospheric pressure chemical ionisation/ fication. Quantitative real-time PCR assays were per- mass spectrometry (LC-APCI–MS) carried out under con- formed using TaqMan Gene Expression Assays (Applied ditions described previously (Marsicano et al. 2002) and Biosystems, Foster City, CA) to quantify mRNA levels for allowing the separations of 2-AG, anandamide, OEA, and Table 1. Major characteristics of patients and control subjects included in this study. Age at sampling in years Group (lowest and highest ages) Gender (M/F) Major clinical characteristics Control subjects (n = 10) 14.1  1.8 (9–29) 6/4 Not relevant Dravet patients (n = 15) 11.2  1.7 (2–26) 9/6 • Age at onset of disease: <12 months • Frequent seizures triggered by fever, light, and/or intense emotions • Frequent motor, speech, and cognitive deficits • Medication: some antiepileptic agents (in most of the cases valproate and/or clobazam, and stiripentol in a few cases), but not taking CBD • Frequent pharmacoresistance CBD, cannabidiol. 2016 | Vol. 4 | Iss. 2 | e00220 ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 4 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. M. Rubio et al. Peripheral Endocannabinoid Signaling and Dravet Syndrome Table 2. List of genes analyzed and Taqman probes used in qRT-PCR Statistics determinations. Data were assessed by analysis of variance followed by the Taqman probe Bonferroni multiple comparison using series of related Gene encoding for reference genes (e.g., CBD targets, endocannabinoid genes, inflam- Glutamate-aspartate transporter (GLAST) Hs00188193_mL mation-related markers, downstream signals). Glutamate transporter type-1 (GLT-1) Hs01102423_mL GABA transporter (GABAT) Hs01104475_mL Serotonin transporter (5HTT) Hs00984349_mL Results Dopamine transporter (DAT) Hs00997364_mL Equilibrative nucleoside transporter (ENT) Hs01085704_gL Characteristics of the cohort of DS patients Adenosine 2A receptor (A2A) Hs00169123_mL investigated 5-hydroxytriptamine 1A receptor (5HT1A) Hs00265014_sL Voltage-dependent calcium Hs00234934_mL All patients recruited for this study (n = 15) had been channel a-1h subunit (CACNA1h) diagnosed of DS with confirmed mutations in the SCN1A Cannabinoid receptor type-1 (CB1) Hs01038522_sL gene, and, in all cases, the disease appeared during the first Cannabinoid receptor type-2 (CB2) Hs00275635_mL 12 months of life. Subject ages ranged from 2 to 26 years, G protein-coupled receptor 55 (GPR55) Hs00995276_mL with an average of 11.2 years at the time of recruitment G protein-coupled receptor 18 (GPR18) Hs01649814_mL Transient receptor potential Hs00218912_mL (Table 1), a range very similar to the currently active GW vanilloid type-1 (TRPV1) Pharma clinical trials with CBD which ranged also from 2 Transient receptor potential Hs00901640_mL to 26 years with a mean age of 10.8 years (www.gw vanilloid type-2 (TRPV2) pharma.com/Epidiolex.aspx). Two-thirds of the subjects Transient receptor potential Hs00175798_mL were male and one-third were female (Table 1). We did ankyrin type-1 (TRPA1) not observe any correlation of data of gene expression or Peroxisome proliferator-activated Hs00234592_mL plasma levels with age or gender (data not shown), possi- receptor-c (PPAR-c) Tumor necrosis factor-a (TNF-a) Hs99999043_mL bly because the number of patients was not sufficiently Interleukin-1b (IL-1b) Hs99999029_mL higher to conduct such correlation analyses. In the case of Cluster of differentiation 70 (CD70) Hs00174297_mL age, it should be noted that we included two patients with Cyclooxygenase-2 (COX-2) Hs00153133_mL an age >20 years (also in controls), but they did not show Inducible nitric oxide synthase (iNOS) Hs01075529_mL any particular differences in the values of different genes Nuclear factor of j-light polypeptide Hs00765730_mL analyzed in comparison with younger patients. The major gene enhancer in B-cells (NFjB) clinical characteristics of these patients consisted of fre- Nuclear factor, erythroid 2-like 2 (Nrf-2) Hs00975691_gL Kelch-like ECH-associated protein 1 (Keap-1) Hs00202227_mL quent seizures (clonic, tonic–clonic, myoclonic) triggered, v-Akt murine thymoma viral Hs00178289_mL among other causes, by fever, light and/or intense emo- oncogene homolog 1 (Akt) tions, as well as significant motor, speech, and cognitive b-arrestin type-1 (ARR1) Hs00244527_mL deficits (Table 1). All patients were taking antiepileptic b-arrestin type-2 (ARR2) Hs01034132_mL medications, in most of the cases valproate and/or cloba- Cytochrome P450, family 1, Hs02382916_sL zam, and in a few cases stiripentol and other alternatives, subfamily B, polypeptide 1 (CYP1B1) but not CBD, although most of the cases exhibited phar- N-arachidonoyl-phosphatidylethanolamine- Hs00419593_mL phospholipase D (NAPE-PLD) macoresistance which is typical of DS patients (Kassa€ı Fatty acid amide hydrolase (FAAH) Hs01038660_mL et al. 2008). Given the existence of this previous medica- Diacylglycerol lipase (DAGL) Hs00391374_mL tion, it is possible that changes we found in lymphocyte Monoacylglycerol lipase (MAGL) Hs00200752_mL gene expression may be related to or at least influenced by Glucuronidase-B (GUSB) Hs00939627_mL such medication, although the differences in the combina- tion of medicines among patients are important, which makes this potential influence poorly probable. PEA. MS detection was carried out in the selected ion monitoring mode using m/z values of 356 and 348 (molecular ion +1 for deuterated and undeuterated anan- Analysis of gene expression in lymphocytes damide), 384.35 and 379.35 (molecular ion +1 for deuter- obtained from DS patients ated and undeuterated 2-AG), 304 and 300 (molecular Lymphocytes from DS patients and control subjects were ion +1 for deuterated and undeuterated PEA), and 328 used first for analysis of genes encoding ion channels or and 326 (molecular ion +1 for deuterated and undeuter- transmitter receptors and transporters that have been ated OEA). The amounts of all endocannabinoids were related to the benefits found with CBD in a number of expressed as pmol/mL of plasma. ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2016 | Vol. 4 | Iss. 2 | e00220 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 5 Peripheral Endocannabinoid Signaling and Dravet Syndrome M. Rubio et al. neurological disorders (Fernandez-Ruiz et al. 2013). The TRPV2 (Fig. 1). We were unable to measure the gene analysis of variance followed by the Bonferroni multiple products for the CB1 and TRPA1 receptors because their comparison confirmed the existence of statistically signifi- expression was below the level of sensitivity (data not cant differences (F(13, 154)=2.872, P < 0.005), which shown). Given that the CB2 receptor has been linked to were evident in the elevated levels of gene expression for the control of inflammatory processes, we then analyzed the CACNA1h in DS patients (Fig. 1). No changes were the expression of some inflammation-related genes (F(11, found for A2A receptors, adenosine transporter, gluta- 149)=3.217, P < 0.01). Those encoding the PPAR-c recep- mate transporters (GLAST and GLT-1), and dopamine tors (which are also activated by CBD) and the cytokines transporter (Fig. 1), although a trend toward a reduction TNF-a and IL-1b (Fig. 2) showed certain trends toward (P = 0.10) was found for the serotonin transporter in DS an increase in DS patients, in particular, in the case of patients (Fig. 1). We were unable to measure the genes IL-1b (P = 0.08), but not in the case of COX-2 (Fig. 2). for the 5HT1A receptor and GABA transporter, because This was in agreement with the upregulation of CB2 their expression was below the level of sensitivity in the receptors, as well as with the elevation in gene expression lymphocytes (data not shown). of CD70 (Fig. 2), a marker of lymphocyte activation. We As regards gene products encoding endocannabinoid- were unable to measure inducible NOS because its expres- related receptors (F(9, 124)=2.596, P < 0.005), we found sion was below the level of sensitivity (data not shown). the CB2 receptor to be significantly elevated in DS We also analyzed intracellular signals downstream the patients, with no changes in GPR55, GPR18, TRPV1, and endocannabinoid signaling, but we did not find any differ- Figure 1. Gene expression for different transmitter transporters (glutamate-aspartate transporter [GLAST], GLT-1, 5HTT, Dopamine transporter (DAT), and ENT) and receptors adenosine 2A receptor (A2A receptor), ion channels (CACNA1h) and endocannabinoid-related receptors (CB2 receptors, GPR55, GPR18, TRPV1, and TRPV2), measured by qRT-PCR in lymphocytes obtained from Dravet syndrome (DS) and control subjects. Values correspond to fold of change over controls and are expressed as means  SEM of 15 DS and 10 controls. Data were assessed by the analysis of variance followed by the Bonferroni multiple comparison. 2016 | Vol. 4 | Iss. 2 | e00220 ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 6 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. M. Rubio et al. Peripheral Endocannabinoid Signaling and Dravet Syndrome Figure 2. Gene expression for peroxisome proliferator-activated receptor-c (PPAR-c) nuclear receptors, the cytokines Tumor necrosis factor-a (TNF-a) and IL-1b, the marker of lymphocyte activation CD70, the proinflammatory enzyme COX-2, different intracellular signaling proteins (NFjB, nuclear factor, erythroid 2-like 2 (Nrf-2), Keap-1, Akt, b-arrestin-1 and -2), and the P450 member CYP1B1, measured by qRT-PCR in lymphocytes obtained from Dravet syndrome (DS) and control subjects. Values correspond to fold of change over controls and are expressed as means  SEM of 15 DS and 10 controls. Data were assessed by the analysis of variance followed by the Bonferroni multiple comparison. ence in transcription factors such as NFjB or Nrf-2, its inhi- measured anandamide, 2-AG, PEA, and OEA in the bitory protein Keap-1, the kinase Akt, and the receptor regu- plasma samples from DS patients and control subjects. latory proteins b-arrestin-1 and -2 (F(11, 148)=0.4891, ns; However, we did not find any changes in these signaling see Fig. 2). We analyzed cytochrome P450 1B1 enzyme iso- lipid derivatives (Fig. 3) in agreement with the data of form, which has been found to be inhibited by CBD gene expression for metabolic enzymes of endocannabi- (Yamaori et al. 2010), although its expression was similar in noids and related N-acylethanolamines in lymphocytes of DS and controls (Fig. 2). Lastly, we analyzed the gene DS patients and controls (Fig. 3). expression of endocannabinoid-related enzymes, for exam- ple, NAPE-PLD, DAGL, FAAH, and MAGL, whose level of Discussion expression, however, was also similar in DS and control sub- jects (F(7, 99) = 0.9307, ns; see Fig. 3). Recent anecdotal and clinical experience with pharmaco- logical preparations based on CBD has led to high expecta- tions for having a new antiepileptic agent with efficacy in Analysis of plasma levels of DS and related syndromes. However, there is no rationale endocannabinoids and related mediators in to explain the presumed efficacy of CBD in these pediatric DS patients syndromes other than the observations derived from stud- In order to determine potential changes in the levels of ies conducted in adult epilepsy, which included both pre- endocannabinoids and related N-acylethanolamines, we clinical studies carried out in seizure models (Hill et al. ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2016 | Vol. 4 | Iss. 2 | e00220 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 7 Peripheral Endocannabinoid Signaling and Dravet Syndrome M. Rubio et al. Figure 3. Gene expression for different endocannabinoid-related enzymes, including NAPE-PLD, fatty acid amide hydrolase (FAAH), diacylglycerol lipase (DAGL), and monoacylglycerol lipase [MAGL], measured by qRT-PCR in lymphocytes obtained from Dravet syndrome (DS) and control subjects, and concentrations of anandamide, 2-arachidonoyl-glycerol, palmitoylethanolamide, and oleylethanolamide measured in the corresponding plasma samples from DS and control subjects. Values correspond to fold of change over controls (gene expression data) or pmol/ mL of plasma (endocannabinoids), and are expressed as means  SEM of 15 DS and 10 controls. Data were assessed by the analysis of variance followed by the Bonferroni multiple comparison. 2012) and the unique clinical trial conducted with CBD in serotonin transporter gene, whose expression tended to adult epileptic patients (Cunha et al. 1980). Because these be lower in DS patients, a result that may be related to syndromes have been proved to be refractory to all cur- some recent evidence indicating that serotonin transporter rently existing antiepileptic medications, there is a need to gene polymorphisms may increase susceptibility to epi- find new medications and also to understand the specific lepsy (Yang et al. 2013). mechanism(s) involved in their effects. Our study repre- Our study also included the analysis of some genes sents a first step toward building the lacking preclinical and encoding proteins of the so-called endocannabinoid sys- clinical understanding of the mechanisms driving CBD tem, although these proteins, except for FAAH, have not activity in these syndromes, together with some recent been particularly associated with the effects of CBD, studies providing incipient evidence on the possible mecha- which may be considered an atypical cannabinoid in rela- nisms underlying CBD effects in DS (Hill et al. 2014; Ian- tion to its activity at the classic endocannabinoid recep- notti et al. 2014; Patel et al. 2014). tors and enzymes. In this case, our objective was to Our first objective was to analyze the changes that the determine the changes that the disease may produce in disease may produce in some pharmacological targets specific elements of the endocannabinoid system, rather (e.g., transmitter receptors and transporters, ion channels) than to find potential targets within this system that may related to the benefits obtained with CBD in a number of explain the antiseizure effects of CBD. We hypothesized neurological disorders (Fernandez-Ruiz et al. 2013), using the existence of a potential dysregulation of the endo- analysis of their gene expression in patient and control cannabinoid signaling system in DS patients, as found in lymphocytes. The only change we found was an increase  other disorders (Massa et al. 2010; Alvaro-Bartolom e and in CACNA1h, which has been proposed as a candidate Garcıa-Sevilla 2013), and reflected in impairments in gene for epilepsy (Frankel 2009). In fact, gain-of-function endocannabinoid levels that may be caused by changes mutations in this gene have been related to infantile in their synthesizing and degrading enzymes, as well as in forms of epilepsy (Frankel 2009), which is in agreement impairments in the density and/or activity of with our results. Another interesting change affected the endocannabinoid receptors. Our major observation was 2016 | Vol. 4 | Iss. 2 | e00220 ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 8 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. M. Rubio et al. Peripheral Endocannabinoid Signaling and Dravet Syndrome an elevated gene expression for the CB2 receptor in DS caution. However, we expect that a similar proinflamma- lymphocytes, although we did not find any change in tory response might also be demonstrated in the brain, endocannabinoid levels in the plasma of DS patients, nor possibly eliciting an elevation of CB2 receptors in glial ele- any change in the gene expression for the major enzymes ments in which they are frequently located (Fernandez- involved in the synthesis and degradation of endo- Ruiz et al. 2007). This question is important in view of cannabinoids. In the brain, the CB2 receptor has been the increasing evidence in support of a role of non-neuro- strongly linked, among others, with the regulation of glial nal components of the CNS, such as glial elements and activation and associated inflammatory events, which may infiltrated peripheral cells, in the pathogenesis of epilepsy support the idea that inflammation could have an influ- (Xu et al. 2013). In support of this, an elevation in the ence in DS, although this possibility has not been fully immunostaining for CB2 receptors in microglial cells has demonstrated in previous studies (Catarino et al. 2011; been recently described in postmortem brain of epilepto- Xu et al. 2013). Elevated CB2 receptor gene expression in genic developmental pathologies (Zurolo et al. 2010), lymphocytes has been also found in other neurological whereas children having autistic features, which also disorders such as multiple sclerosis (Sanchez L opez et al. appear in DS, showed elevated levels of this receptor in 2015) and autism (Siniscalco et al. 2013), although the lymphocytes (Siniscalco et al. 2013) as in our study. A issue has not been investigated, to the best of our knowl- previous study described increased levels of proinflamma- edge, in epilepsy. It is interesting to note that in the case tory cytokines in these children (Molloy et al. 2006), as of multiple sclerosis, the elevation found in lymphocytes was also observed here. All these data point to the poten- (Sanchez L opez et al. 2015) recapitulated a similar tial role that the activation of CB2 receptors may have in response measured in CNS structures (Fernandez-Ruiz these disorders, including DS, and this potential would et al. 2007) and was reversed by progressive interferon support the possibility to develop cannabinoid-based therapy (Sanchez L opez et al. 2015). This allows lympho- therapies with more benefits for the treatment of DS than cyte CB2 expression to be used as a peripheral biomarker just an antiepileptic activity. For example, they may be for monitoring treatment efficacy, something that could potentially effective in delaying/reducing the occurrence be of interest also for DS. of motor, speech, and cognitive deficits, which are also In addition to the elevation of CB2 receptor gene important disabling consequences of this pathology, given expression, we found that other inflammation-related their well-known anti-inflammatory and neuroprotective markers, for example, PPAR-c receptors and proinflam- properties largely demonstrated in preclinical models of matory cytokines, also showed certain trends toward an neurodegenerative disorders (Iuvone et al. 2009; Fernan- increase, although they did not reach statistical signifi- dez-Ruiz et al. 2011). All these questions are currently cance. These trends were due to subsets of patients having under investigation and will benefit from the availability significantly higher expression levels than the mean of the of mouse models of DS (Oakley et al. 2011). control group. We examined whether these subsets of In summary, our data showed changes in a few potential patients exhibited some particular characteristics in rela- CBD targets, for example, the serotonin transporter and, in tion to their clinical data (e.g., age, gender, seizure fre- particular, the CACNAC1h, in DS patients. They also quency, type of medication), but we did not find any proved the existence of an upregulation of CB2 receptors in positive correlation, possibly also due to the relatively less lymphocytes, associated with the activation of these cells, in number of subjects employed in this study. An interesting DS patients, similar to the responses found with this recep- observation was that these responses were paralleled by tor in neuroinflammatory disorders. In the case that such an increase in gene expression for CD70 (the Ki24 anti- response also occurs in the brain and that CB2 receptors are gen), a surface receptor which serves as a marker for lym- involved in the physiopathology of DS, this finding would phocyte proliferation and activation (Borst et al. 2005; extend the possible cannabinoid-based therapies for this Shipkova and Wieland 2012). Therefore, we may hypoth- disease, not only for the reduction of seizures, but particu- esize that lymphocytes are activated in DS patients, which larly for the control of inflammation and other responses may presumably cause the elevation of CB2 receptors. In underlying cognitive and motor deficits. Importantly, our this respect, it is important to note that our data (ele- study uncovers a dysregulation of the endocannabinoid sys- vated CB2 receptor and CD70 gene expression) may be tem in patients with DS that might shed light on the biol- also interpreted as a mere reflection of specific changes ogy of this drug refractory syndrome. occurring in the immune system and not necessarily occurring in the brain, a fact that may be normal given Acknowledgements the frequent seizure activity experienced by these patients which should trigger peripheral inflammation-related This work was supported by grants from CIBERNED responses. Therefore, our data should be taken with (CB06/05/0089), MINECO (SAF2012/39173), and CAM ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2016 | Vol. 4 | Iss. 2 | e00220 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. 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