Analysis of endocannabinoid signaling elements and related proteins in lymphocytes of patients with Dravet syndrome

Abstract

Cannabidiol (CBD) reduces seizures in childhood epilepsy syndromes including Dravet syndrome (DS). A formulation of CBD has obtained orphan drug designation for these syndromes and clinical trials are currently underway. The mechanism responsible for CBD effects is not known, although it could involve targets sensitive to CBD in other neurological disorders. We believe of interest to investigate whether these potential targets are altered in DS, in particular whether the endocannabinoid system is dysregulated. To this end, lymphocytes from patients and controls were used for analysis of gene expression of transmitter receptors and transporters, ion channels, and enzymes associated with CBD effects, as well as endocannabinoid genes. Plasma endocannabinoid levels were also analyzed. There were no differences between DS patients and controls in most of the CBD targets analyzed, except an increase in the voltage-dependent calcium channel α-1h subunit. We also found that cannabinoid type-2 (CB 2) receptor gene expression was elevated in DS patients, with no changes in other endocannabinoid-related receptors and enzymes, as well as in plasma levels of endocannabinoids. Such elevation was paralleled by an increase in CD70, a marker of lymphocyte activation, and certain trends in inflammation-related proteins (e.g., peroxisome proliferator-activated receptor-γ receptors, cytokines). In conclusion, together with changes in the voltage-dependent calcium channel α-1h subunit, we found an upregulation of CB 2 receptors, associated with an activation of lymphocytes and changes in inflammation-related genes, in DS patients. Such changes were also reported in inflammatory disorders and may indirectly support the occurrence of a potential dysregulation of the endocannabinoid system in the brain.

Keywords: Dravet syndrome; endocannabinoid signaling; endocannabinoids; lymphocytes.

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 (CB ₂ 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.

Figure 2

Gene expression for peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) nuclear receptors, the cytokines Tumor necrosis factor‐α (TNF‐α) and IL‐1β, the marker of lymphocyte activation CD70, the proinflammatory enzyme COX‐2, different intracellular signaling proteins (NF κB, nuclear factor, erythroid 2‐like 2 (Nrf‐2), Keap‐1, Akt, β‐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.

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.