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Abstract

Background

The endocannabinoid system (ECS) is one of the key endogenous systems regulating pain. Preclinical and clinical evidence suggests a dysregulation of the ECS in patients with migraine. The present study aimed to characterize the main ECS components in patients with chronic migraine and medication overuse headache (MOH) and episodic migraine (EM) at the gene expression level compared to healthy controls (HC) and to correlate the findings with psychopathological scales.

Methods

In this cross-sectional study, consecutive patients with a diagnosis of EM and MOH were enrolled. Fatty acid amide hydrolase (FAAH) was assayed through quantitative enzyme-linked immunosorbent assay kits. The gene expression of ECS components (FAAH, N-arachidonoyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and N-acylethanolamine acid amidase (NAAA) enzymes, cannabinoid (CB) receptors, CB₁ and CB₂, transient receptor potential vanilloid type 1 (TRPV1) and peroxisome proliferator-activated receptor (PPAR)ɑ receptors was assayed in peripheral blood mononuclear cells through a real-time quantitative PCR. Clinical data including Migraine Disability Assessment (MIDAS) and Headache Impact Test-6 (HIT-6) were collected. Psychopathological status was assessed through the Hamilton Anxiety Rating Scale (HAM-A), the Hamilton Rating Scale for Depression (HAM-D) and the Toronto Alexithymia Scale (TAS-20).

Results

The study included 31 patients (15 with EM, 16 with MOH) and 14 HC. The gene expression of FAAH, an enzyme involved in the degradation of the main endocannabinoid, was significantly lower in MOH patients (0.0002 ± 0.0002) than in EM patients (0.0008 ± 0.0006) (p = 0.005). There were no significant differences in gene expression among EM, MOH and HC groups for NAPE-PLD, NAAA, CB₁, CB₂, TRPV1 and PPARɑ. The levels of FAAH protein expression were significantly higher in MOH (2.9517 ± 2.2006 pg/μg) compared to EM patients (0.9225 ± 0.6878 pg/μg) (p = 0.025). In the clinical group (EM and MOH), we found a significant negative correlation between FAAH gene expression and FAAH enzyme protein (p = 0.014); FAAH gene expression negatively correlated with HIT-6 (p = 0.003) and MIDAS scores (p = 0.048), as well with all psychopathological scales, in more detail with TAS-20 (p = 0.029), HAM-A (p = 0.040) and HAM-D (p = 0.028).

Conclusions

FAAH undergoes specific alterations in patients with MOH at gene expression levels, suggesting its potential as a blood biomarker for this condition. FAAH gene expression is possibly related to psychiatric comorbidities in migraine patients.

Graphical abstract

This is a visual representation of the abstract.

Keywords

anandamide endocannabinoid system FAAH medication overuse headache migraine

Introduction

Migraine is a disabling primary headache disorder affecting approximately 15% of the population (1). Medication-overuse headache (MOH) is a secondary headache disorder resulting from frequent intake of acute medications (2). Medication-overuse headache pathogenesis may involve progressive changes in synapses, neurons, neurotransmitters, connectivity and pain control due to the repetition of external and internal stimuli (2). In its complexity, MOH is commonly associated with psychiatric comorbidities such as anxiety disorders and major depression (3). The prevalence of psychiatric comorbidities in MOH exceeds that observed in certain chronic primary headache disorders, such as chronic migraine (CM) and chronic tension-type headache (4,5).

The endocannabinoid system (ECS) is a distinctive and complex signaling network widely distributed throughout the body, where it regulates various processes, including pain perception, inflammation, and neurotransmitter release and function. The ECS has also been implicated in the etiopathogenesis of various psychiatric disorders, including anxiety and depression (6).

The fundamental components of the ECS are two bioactive lipids, also known as endocannabinoids: N-arachidonoylethanolamine or anandamide (AEA) and 2-arachidonoylglycerol (7). Both molecules are released in response to specific stimuli and exert their functions through the two primary cannabinoid (CB) receptors, CB₁ and CB₂ (7). Furthermore, the synthesis and degradation of these lipids are finely regulated by their respective metabolic enzymes (7). Of note, AEA is generated upon inflammation and exerts its biological activity not only by predominantly binding to CB₁ and CB₂, but also to additional non-cannabinoid receptors such as the transient receptor potential vanilloid type 1 (TRPV1) and the nuclear peroxisome proliferator-activated receptors (PPARs). The activity of AEA at its receptor targets is controlled by the endogenous tone of this lipid, which in turn is controlled by metabolic enzymes (7). Anandamide is principally biosynthesized from N-arachidonoyl phosphatidylethanolamines (NAPEs) via a NAPE-specific phospholipase D (NAPE-PLD); then, it is degraded by fatty acid amide hydrolase (FAAH), and to a lower extent by the lysosomal N-acylethanolamine acid amidase (NAAA) (7). In this context, inhibition of AEA degradation by FAAH emerged as a promising therapeutic target for migraine pain because it is probably related to increased availability of the endocannabinoid at the site of stimulation (e.g. trigeminal ganglion and/or meninges), which results in a prolonged action (8 –12).

Clinical findings showed dysfunction of the ECS in patients with episodic migraine (EM), where an increased FAAH activity, consistent with reduced AEA concentration, was documented in platelets (13). Furthermore, studies demonstrated lower levels of endocannabinoids in patients with CM and MOH (14,15). More recently, the transcriptional changes in ECS components were investigated in patients with migraine. Remarkably, gene (mRNA) and protein expression of FAAH was found to decrease, whereas that of CB₁ and CB₂ increased in peripheral blood mononuclear cells (PBMCs) of both MOH and EM patients compared to control subjects (16). A correlation was also found between some ECS components and clinical features of migraine, such as the number of monthly migraine days (16). However, to date, correlations between ECS components and psychiatric/psychological aspects in patients with migraine have not been investigated yet.

The present study aimed to further characterize the main AEA-related ECS components in MOH patients at the gene expression level. In addition, FAAH gene and protein levels were assayed in MOH patients compared to EM and healthy controls (HC). Correlation between ECS components, clinical features of migraine and depression, anxiety and alexithymia were also explored.

Methods

Setting and participants

This cross-sectional study conducted in a tertiary headache center, enrolled consecutive patients with a diagnosis of chronic migraine and MOH (MOH group) or EM, according to The International Classification of Headache Disorders 3rd edition criteria (ICHD-3) (17), and age-matched HC. Medication overuse headache was defined, according to ICHD-3 criteria, as (A) headache occurring on ≥15 days/month in a patient with a pre-existing headache disorder; (B) regular overuse for >3 months of one or more drugs that can be taken for acute and/or symptomatic treatment of headache; and (C) not better accounted for by another ICHD-3 diagnosis (17). The inclusion period ranged from February 2022 to September 2022. We included patients >18 years old who provided their informed consent. Exclusion criteria included refusal to give informed consent, prior diagnosis of cognitive deterioration or Montreal Cognitive Assessment (MoCA) score <25 (18), pregnancy, psychiatric or relevant systemic diseases (moderate to severe cardiac, renal, and hepatic failure), use of illicit drugs including cannabis and its derivatives, patients not on a stable dosage and regimen of preventive treatments for at least three months prior to blood collection.

All healthy control subjects were selected from staff members and were interviewed directly by a neurologist and headache specialist, who had no previous diagnosis of primary or secondary headache disorders.

The study adheres to the ethical principles outlined in the 1975 Declaration of Helsinki. It received approval from the Ethics Committee of Università Cattolica del Sacro Cuore in Rome.

Assessment of migraine

A neurologist and headache specialist assessed migraine features using a structured interview. Information was gathered about socio-demographic data, age at onset of the headache disorder, presence of aura, duration of attacks, frequency of headache (monthly headache days (MHDs) and monthly migraine days (MMDs)), history of acute and preventive treatments and acute medication intake, including the absolute number of analgesics (AMNs). Additionally, the severity and the headache-related disability were assessed through the Headache Impact Test-6 (HIT-6) and the Migraine Disability Assessment (MIDAS) questionnaire.

Concomitant psychopathological conditions were assessed through different scales: the Hamilton Anxiety Rating Scale (HAM-A), the Hamilton Rating Scale for Depression (HAM-D) and the Toronto Alexithymia Scale (TAS-20). The HAM-A (19) is a clinician-rated scale that assesses the presence and entity of anxiety. The HAM-A scores are categorized as mild (8–14), moderate (15–23) and severe ≥ 24 anxiety (20). The HAM-D (21) evaluates the severity of depression. A score in the range 10–13 is suggestive of mild depression, 14–17 is suggestive of of moderate and >17 is suggestive of of severe depression (22). The TAS-20 is a 20 multiple-choice self-administered scale that measures difficulty in identifying and describing feelings, namely alexithymia. The cut-off for alexithymia on the TAS-20 total score is >61/100 (23).

Subjects were also screened for cognitive impairment with the MoCA (18).

Blood sample collection and analysis of ECS components

Blood samples (30 ml per subject) drowned from the antecubital vein of patients were collected into heparinized sterile tubes.

Peripheral blood cell isolation

After venous puncture, peripheral blood mononuclear cells (PBMCs) were isolated from HC, EM, and MOH subjects using a density gradient separation with Ficoll-Hypaque (Amersham Biosciences, Chicago, IL, USA) in accordance to standard procedures (24). Specifically, 10 ml of peripheral blood was diluted 1:1 with sterile phosphate-bufered saline (PBS) and carefully layered on top of 15 ml of Ficoll. The sample was then centrifuged for 30 minutes at 1800 rpm (652 g) at 4°C in a swing-out bucket with no brake. The buffy coat with the PBMCs formed at the interface between the Ficoll and plasma was carefully collected with a sterile Pasteur pipette and washed twice in sterile PBS for 10 minutes at 4°C. Subsequently, the PBMCs were resuspended in RPMI 1640 complete medium supplemented with 10% (v/v) heat-inactivated human serum before additional processing.

Quantitative reverse transcriptase-PCR (qRT-PCR)

Total RNA from PBMCs was extracted with a ReliaPrep RNA Miniprep System kit (Promega, Madison, WI, USA). SuperScript IV VILO Reverse Transcriptase (Invitrogen, Waltham, MA, USA) was employed for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on a StepOne Real-Time PCR System sequence detector (Applied Biosystems, Foster City, CA, USA) with Applied Biosystems predesigned TaqMan Gene Expression Assays. The probes used (Applied Biosystems, assay identification numbers in parentheses) were: CB₁ receptor (Hs01038522_s1), CB₂ receptor (Hs05019229_s1), TRPV1 (Hs00218912_m1), peroxisome proliferator-activated receptor alpha (PPARɑ) (Hs00947536_m1), NAPE-PLD (Hs00419593_m1), FAAH (Hs1038664_m1) and N-acylethanolamine acid amidase (NAAA) (Hs00384501_m1). For each sample, the abundance of each gene was evaluated with respect to beta-actin expression (Hs01060665_g1).

Enzyme-linked immunosorbent assay (ELISA)

The protein expression of FAAH was measured through quantitative ELISA kits in a subset of subjects from PBMCs (Novus Biologicals, LLC, Centennial, CO, USA). The detection relies on a conventional sandwich ELISA, and absorbance was read between 405/420 nm (assay sensitivity 46.88 pg/ml) on a VariosScan Flash (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions.

Statistical analysis

The sample size was determined using G*Power software (http://www.gpower.hhu.de), based on an a priori power analysis for one-way analysis of variance (ANOVA). Data on FAAH gene expression levels (mean ± SD: 2.86 ± 1.99 for HC, 1.40 ± 0.90 for EM and 0.43 ± 0.22 for MOH subjects) were derived from a previous study (16). The effect size (Cohen's f = 0.70) was calculated from these values, with a significance level (α) of 0.05 and a power (1 – β) of 0.80, yielding a total sample size of 24 participants (eight per group). No imputation was made for missing data; the analyses were performed based on the available sample. Demographical and clinical data are reported using descriptive statistics. Numerical variables are reported as the mean ± SD or median and interquartile range, whereas categorical variables are reported as absolute numbers (n) and percentages (%) and compared using the chi-squared test. The normal distribution of each numerical variable was assessed using the Kolmogorov–Smirnov test and performed parametric or non-parametric analyses in accordance. Parametric or non-parametric ANOVA (Kruskal–Wallis test) was performed to compare the three groups (EM, MOH and HC) on each ECS component according to the distribution. Bonferroni's correction was applied to adjust for multiple comparisons between groups. Comparisons between the two clinical groups (EM and MOH) were performed with the Mann–Whitney U-test. Correlations between FAAH gene expression and FAAH levels were computed by two-tailed Pearson's correlation (r) or Spearman's rank correlation (ρ) coefficients according to the distribution of the variables.

Moreover, correlations were also performed between endocannabinoids and clinical variables and psychopathological scales. To assess the hypothesized negative correlation between ECS components and depression, anxiety, and alexithymia and clinical features of migraine, based on data presented in the literature (16,25), we employed Pearson's correlation or Spearman's rank correlation coefficients as appropriate using a one-tailed test. p < 0.05 was considered statistically significant. The statistical analysis was performed with SPSS, version 21.0 (IBM Corp., Armonk, NY, USA) and Jamovi, version 2.3.26.0 (https://www.jamovi.org).

Results

Sample characteristics

The sample included 31 patients with migraine and 14 HCs. In particular, it enrolled 15 patients with EM with a mean ± SD age of 31.33 ± 10.55 years and 16 MOH patients with a mean ± SD age of 38.88 ± 13.72 years. The HC were 14 age-matched subjects with a mean ± SD age of 29.21 ± 9.43 years. The clinical and demographic features of the sample are shown in Table 1.

Table 1.

Clinical and demographical features of the cohort.

	HC (n = 14)		EM (n = 15)		MOH (n = 16)
							p	Pairwise comparisons
								HC vs EM	HC vs MOH	EM vs MOH
								p _Bonf	p _Bonf	p _Bonf
Age (mean, SD)	29.21	9.43	31.33	10.55	38.88	13.71	0.308 #	NS	NS	NS
Sex, male/female, n (%)	7 (50.0%)/7 (50.0%)		8 (53.3%)/7(46.7%)		4 (25.0%)/12 (75.0%)		0.217 °	NS	NS	NS
HAM-A (mean, SD)	11.2	10.2	9.6	10.1	16.1	10.5	0.132 §	NS	NS	NS
HAM-D (mean, SD)	12.7	9.2	11.3	8.0	13.8	8.6	0.658 §	NS	NS	NS
TAS-20 (mean, SD)	52.4	9.7	52.1	9.5	57.4	9.0	0.313 §	NS	NS	NS
Duration of migraine (years)	NA	NA	11.00	8.10	21.18	15.54	0.165 §
MHDs (mean, SD)	NA	NA	2.69	1.93	16.56	4.23	<0.001 §
MMDs (mean, SD)	NA	NA	2.21	1.48	14.63	4.94	<0.001 §
AMNs (mean, SD)	NA	NA	3.50	3.07	29.00	36.80	<0.001 §
HIT-6	NA	NA	54.38	8.01	65.08	5.27	0.002 §
MIDAS	NA	NA	13.23	17.58	61.77	43.25	0.001 §

Abbreviations: AMNs = absolute number of analgesics; EM = episodic migraine; HAM-A = Hamilton Anxiety Rating Scale; HAM-D = Hamilton Rating Scale for Depression; HC = healthy controls; HIT-6 = Headache Impact Test-6; MHDs = monthly headache days; MIDAS = Migraine Disability Assessment; MMD = monthly migraine days; MOH = medication-overuse headache; NA = not applicable; NS = not significant; pBonf = p-value adjusted for Bonferroni’s correction; TAS-20 = Toronto Alexithymia Scale. #Non-parametric Kruskal–Wallis test. °Chi-squared test. §Mann–Whitney U-test. The bold characters indicate statistical significance.

The mean ± SD MHDs were 2.69 ± 1.93 for the EM group and 16.56 ± 4.23 for the MOH group, and the MMDs were 2.21 ± 1.48 for the EM group and 14.63 ± 4.94 for the MOH group. The mean ± SD AMNs were 3.50 ± 3.07 for the EM group and 29.00 ± 36.80 for the MOH group. In the EM group, one patient (6.67%) was taking a preventive treatment, whereas, in the MOH group, seven patients (43.75%) were taking a preventive treatment.

No significant differences were observed in the psychopathological scales (HAM-A, HAM-D and TAS-20) across the three groups (Table 1).

Differential expression of key players in AEA signaling in PBMCs of HC, EM and MOH groups

First, the gene expression of key receptors and enzymes involved in the synthesis and degradation of AEA were analyzed. By using real-time PCR, FAAH gene expression was found to be significantly lower in MOH patients (0.0002 ± 0.0002) compared to EM patients (0.0008 ± 0.0006) (p = 0.005). However, there were no significant differences in gene expression among the EM, MOH and HC groups for NAPE-PLD and NAAA enzymes, as well as CB₁, CB₂, TRPV1 and PPARɑ receptors (Figure 1 and Table 2).

Figure 1.

Gene expression of N-arachidonoylethanolamine or anandamide (AEA)-related endocannabinoid system (ECS) elements and protein expression of fatty acid amide hydrolase (FAAH) in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), episodic migraine (EM) and medication-overuse headache (MOH) patients. The bars represent the median and interquartile range. The statistical comparison between groups was performed using the Kruskal–Wallis test. Protein expression of FAAH was assayed in a subset of subjects including five HC, seven EM and six MOH. CB₁ = cannabinoid receptor 1; CB₂ = cannabinoid receptor 2; NAAA = N-acylethanolamine acid amidase; NAPE-PLD = N-arachidonoyl-phosphatidylethanolamine-specific phospholipase D; PPARɑ = peroxisome proliferator-activated receptor alpha; TRPV1 = transient receptor potential vanilloid type 1. *: statistically significant.

Table 2.

Results of gene expression of the endocannabinoid system components and quantification of fatty acid amide hydrolase (FAAH) protein expression from peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), episodic migraine (EM) and medication-overuse headache (MOH) groups.

	HC		EM		MOH
	HC		EM		MOH		p	Pairwise comparisons
								HC vs EM	HC vs MOH	EM vs MOH
	Mean	SD	Mean	SD	Mean	SD		p _Bonf	p _Bonf	p _Bonf
FAAH protein (pg/μg)	0.8980	0.6128	0.9225	0.6878	2.9517	2.2006	0.025*	NS	NS	0.039
FAAH	0.0004	0.0004	0.0008	0.0006	0.0002	0.0002	0.005*	NS	NS	0.005
NAPE-PLD	0.0012	0.0012	0.0014	0.0010	0.0008	0.0008	0.707#	NS	NS	NS
NAAA	0.0062	0.0044	0.0064	0.0048	0.0058	0.0039	0.920*	NS	NS	NS
CB₁	0.0024	0.0028	0.0009	0.0028	0.0035	0.0028	0.210#	NS	NS	NS
CB₂	0.0101	0.0081	0.0101	0.0096	0.0101	0.0418	0.320#	NS	NS	NS
TRPV1	0.0005	0.0004	0.0004	0.0004	0.0003	0.0002	0.263*	NS	NS	NS
PPARɑ	0.0038	0.0028	0.0035	0.0031	0.0038	0.0029	0.962*	NS	NS	NS

Abbreviations: CB1 = cannabinoid receptor 1; CB2 = cannabinoid receptor 2; EM = episodic migraine; FAAH = fatty acid amide hydrolase; HC = healthy controls; MOH = medication-overuse headache; NS = not significant; NAAA = N-acylethanolamine acid amidase; NAPE-PLD = N-arachidonoyl phosphatidylethanolamine-specific phospholipase D; PPARα = peroxisome proliferator-activated receptor α; pBonf = p-value adjusted for Bonferroni’s correction. #Non-parametric Kruskal–Wallis test. *Analysis of variance. The bold characters indicate statistical significance.

Next, the levels of FAAH protein expression in a subgroup of subjects (HC, n = 5; EM, n = 7; and MOH, n = 6) were quantified, with significantly higher values of FAAH in MOH patients (2.9517 ± 2.2006 pg/μg) compared to EM patients (0.9225 ± 0.6878 pg/μg) (p = 0.025) (Table 2).

In the clinical group (EM and MOH), we also found a significant negative correlation between FAAH gene expression and FAAH enzyme protein (Pearson’ r = −0.660, p = 0.014).

Correlations between ECS components, clinical features of migraine and psychopathological scales

In the whole sample, correlations between ECS components are reported in the supplementary material, Table S1.

In the clinical group (EM and MOH), FAAH gene expression negatively correlated with HIT-6 score (Spearman's ρ = −0.556, p = 0.003) and MIDAS score (Spearman's ρ = −0.356, p = 0.048). Gene expression of NAAA was negatively correlated with MIDAS score (Spearman's ρ = −0.353, p = 0.042).

FAAH gene expression was negatively correlated with all psychopathological scales, in more detail with TAS-20 (Spearman's ρ = −0.375, p = 0.029), HAM-A (Spearman's ρ = −0.344, p = 0.040) and HAM-D (Spearman's ρ = −0.371, p = 0.028). Moreover, TAS-20 resulted negatively correlated with NAAA (Spearman's ρ = −0.373, p = 0.025), TRPV1 (Spearman's ρ = −0.447, p = 0.011), NAPE-PLD (Spearman's ρ=−0.405, p = 0.020) and PPARɑ (Spearman's ρ = −0.320, p = 0.045). Finally, NAAA gene expression was also negatively correlated with the HAM-D score (Spearman's ρ = −0.433, p = 0.009).

All the correlations and coefficients are summarized in Table 3, and scatterplots of all statistically significant correlations are shown in Figure 2.

Figure 2.

Scatterplots of all statistically significant correlations between endocannabinoid system (ECS) components with respect to gene expression, Headache Impact Test-6 and Migraine Disability Assessment scores and psychopathological scales. FAAH = fatty acid amide hydrolase; HAM-A = Hamilton Anxiety Rating Scale; HAM-D = Hamilton Rating Scale for Depression; HIT-6 = Headache Impact Test-6; MIDAS = Migraine Disability Assessment; NAAA = N-acylethanolamine acid amidase; NAPE-PLD = N-arachidonoyl phosphatidylethanolamine-specific phospholipase D; PPARɑ = peroxisome proliferator-activated receptor alpha; TAS-20 = Toronto Alexithymia Scale; TRPV1 = transient receptor potential vanilloid type 1.

Table 3.

Correlations of endocannabinoid system gene expression with clinical features of migraine for the clinical groups (episodic migraine and medication-overuse headache), using Spearman's rank correlation coefficient using a one-tailed test.

		FAAH	NAAA	PPARɑ	CB₁	CB₂	TRPV1	NAPE-PLD
TAS-20	Spearman's ρ	−0.375	−0.373	−0.320	0.247	0.011	−0.447	−0.405
TAS-20	p-value	0.029	0.025	0.045	0.872	0.521	0.011	0.020
HAM-D	Spearman's ρ	−0.344	−0.433	−0.186	−0.059	−0.189	−0.128	−0.291
HAM-D	p-value	0.040	0.009	0.163	0.391	0.188	0.262	0.071
HAM-A	Spearman's ρ	−0.371	−0.227	−0.093	0.033	−0.269	0.008	−0.085
HAM-A	p-value	0.028	0.119	0.312	0.561	0.102	0.515	0.336
HIT-6	Spearman's ρ	−0.556	−0.310	0.082	0.338	−0.060	0.065	−0.080
HIT-6	p-value	0.003	0.066	0.655	0.933	0.401	0.618	0.356
MIDAS	Spearman's ρ	−0.356	−0.353	0.098	0.444	0.054	0.121	−0.147
MIDAS	p-value	0.048	0.042	0.683	0.978	0.589	0.714	0.247
MMDs	Spearman's ρ	−0.269	0.0809	0.1338	0.4342	0.3588	0.2045	0.1461
MMDs	p-value	0.113	0.646	0.738	0.972	0.94	0.819	0.742
MHDs	Spearman's ρ	−0.280	−0.084	0.148	0.361	0.191	0.256	−0.006
MHDs	p-value	0.083	0.335	0.779	0.955	0.809	0.896	0.489
AMNs	Spearman's ρ	−0.240	0.042	0.271	0.470	0.443	0.135	0.073
AMNs	p-value	0.168	0.570	0.882	0.975	0.967	0.703	0.617

Abbreviations: AMN = absolute number of analgesics; CB₁ = cannabinoid receptor 1; CB₂ = cannabinoid receptor 2; FAAH = fatty acid amide hydrolase; HAM-A = Hamilton Anxiety Rating Scale; HAM-D = Hamilton Rating Scale for Depression; HIT-6 = Headache Impact Test-6; MHD = monthly headache days; MIDAS = Migraine Disability Assessment; MMDs = monthly migraine days; NAAA = N-acylethanolamine acid amidase; NAPE-PLD = N-arachidonoyl phosphatidylethanolamine-specific phospholipase D; PPARα = peroxisome proliferator-activated receptor alpha; TAS-20 = Toronto Alexithymia Scale; TRPV1 = transient receptor potential vanilloid type 1. The bold characters indicate statistical significance.

Discussion

The present study showed an alteration of the main AEA-related ECS components in PBMCs isolated from patients with MOH. In particular, MOH patients exhibited lower FAAH gene expression and higher FAAH protein levels compared to EM patients. Overall, across all clinical groups, there was an inverse correlation between FAAH protein expression and FAAH gene expression. Instead, we found no other significant difference in gene expression of the other ECS components under investigation, including all AEA-binding receptors and additional metabolic enzymes.

Fatty acid amide hydrolase is a key component in the ECS. Several investigations support the idea that FAAH plays a role in regulating pain perception by influencing the endogenous levels of endocannabinoids (8 –10,26,27). For example, studies on episodic migraine patients showed an increased FAAH activity in platelets, consistent with reduced AEA concentration and upregulation of CB₁secondary to an endocannabinoid deficiency (13,28).

Some investigations have been undertaken in patients with CM and MOH, demonstrating a deficiency of endocannabinoids in platelets and cerebrospinal fluid, which is more pronounced in MOH patients (15,29). To the best of our knowledge, there is only one study that examined the gene expression of ECS components in people with migraine, showing that FAAH gene expression was lower in MOH and EM patients compared to HC; yet, FAAH protein expression was not assayed in the same study (16). Furthermore, EM and MOH subjects showed higher CB₁ and CB₂ gene and protein expression compared to healthy controls (16).

In the present study, we confirmed a significant reduction in FAAH gene expression among MOH patients compared to patients with EM (16). Previous studies investigating FAAH protein levels in MOH patients did not yield consistent findings, and the difficulty in comparing these earlier results with ours appears to be a result of differences in techniques and cellular substrates (14). At any rate, our study supports the observation that in MOH patients, there is an increase in FAAH protein consistent with a larger degradation of the FAAH substrate AEA. Furthermore, the downregulation of FAAH gene expression could be a compensatory mechanism to counteract the effects of chronic increases in FAAH protein levels in MOH patients. It could be speculated that this regulatory mechanism aimed to preserve the availability of AEA.

However, aside from FAAH, no other significant differences in the expression of ECS component genes were observed across the three groups, in contrast to the findings of Greco et al. (16). Further studies are required to determine whether these results were influenced by the small sample size or whether FAAH may be a more specific and sensitive biomarker for migraine, particularly in MOH.

Finally, although all psychopathological scale values were elevated in the MOH group, we did not find significant differences between the three groups, likely due to the small sample size. However, we found a significant negative correlation between FAAH gene expression and both HIT-6 and MIDAS scores. These results suggest an association between ECS gene expression and migraine-related disability and severity. Fatty acid amide hydrolase gene expression was also negatively correlated with HAM-A, HAM-D and TAS-20 scores, indicating that patients with a downregulation of this enzyme, who were the MOH patients in the present study, exhibit higher levels of anxiety, depression, and alexithymia.

Previous reports have already demonstrated a higher psychiatric comorbidity burden in MOH patients, and it could be speculated that the ECS may be involved in the pathogenesis of these disturbances in migraine patients (2). Interestingly, the alexithymia score was inversely correlated with most ECS gene expression, suggesting that lower ECS gene expression is associated with higher levels of alexithymia. Alexithymia, namely the difficulty in identifying and describing one's own emotions, is a trait consisting of a disorder of negative affect regulation, which is dysregulated in patients with migraine, especially in those with MOH (30 –32). Dysfunction of the anterior cingulate cortex has been proposed to be one of the main mechanisms of alexithymia. Indeed, CB1 receptors are highly expressed in brain structures such as the amygdala, hippocampus and anterior cingulate cortex, which are crucial regions in the regulation of emotional responses, prompting the speculation that a reduction in the endocannabinoid tone may be associated with alexithymia in patients with migraine (33).

In the present study, a correlation between ECS components and the number of symptomatic drugs was not demonstrated, possibly suggesting that the ECS dysregulation found in patients with MOH may not be primarily related to chronic exposure to acute medications commonly used for migraine, such as non-steroidal anti-inflammatory drugs, instead suggested by other studies (34). To corroborate this hypothesis, previous findings showed a dysfunction in the ECS in patients with CM without medication overuse, suggesting a potential connection with chronic headache independent of medication intake; furthermore, in patients with MOH, withdrawal from medications was associated with a significant reduction in FAAH activity in the platelets in parallel with the improvement in clinical parameters (15,29,35).

The findings from the present study, combined with previous evidence on endocannabinoid dysfunction in individuals with migraine, prompt speculation regarding the pathophysiological aspects of chronic migraine and MOH. The downregulation of the ECS gene expression could contribute to abnormal modulation of brain nociceptive systems, resulting in central sensitization, potentially explaining the transformation of migraine from episodic to chronic. Moreover, because the ECS also plays a role in drug addiction, it is plausible that an impaired endocannabinoid tone might influence the compulsive reward-seeking behavior implicated in MOH development (36,37).

Currently, the recommended strategy for treating medication overuse is to inform and educate the patients about the condition, withdraw the overused medication and start a preventive treatment (2). Nevertheless, clinical and preclinical observations support the hypothesis that the modulation of the metabolic pathways of the ECS could pave the way for innovative migraine treatments, with a particular focus on the FAAH enzyme as a promising target (8 –12).

One strength of the present study is the extremely careful selection of the patients included and the technique used. Indeed, we assessed ECS components from PBMCs that share similarities with neuronal biochemical environments (38). Furthermore, it is noteworthy that peripheral changes in ECS components detected in PBMCs appear to mirror central dysfunction of the ECS in several neurological diseases (39). Additionally, this is the first study to correlate FAAH gene expression with FAAH levels and to provide correlations between the ECS components and psychological scales, including depression, anxiety and alexithymia.

The study has some limitations. First, the sample size was relatively small, warranting further investigation in larger cohorts to validate these findings. Furthermore, the cross-sectional design of the study does not allow the establishment of causality between the altered ECS components and medication-overuse status. Lastly, circulating levels of endocannabinoids were not assayed in the present study. This decision was motivated by the fact that these levels are influenced by several physiological factors, including physical exercise, stress and circadian rhythm, making them highly unstable in biological samples and not suitable as potential biomarkers.

Conclusions

The ECS plays a critical role in the pathogenesis of migraine, and, in particular, its dysfunctions may have significant implications for the pathogenesis of MOH and the associated psychiatric comorbidities and psychopathological traits. Fatty acid amide hydrolase, in particular, appears to undergo specific alterations in patients with MOH. Clinical implications

The endocannabinoid system is dysregulated in patients with migraine, playing a critical role in its pathogenesis

In patients with MOH, there is an increased activity of the enzyme FAAH, which is involved in the degradation of the main endocannabinoid, and a significant downregulation of FAAH gene expression.

Dysfunctions of the endocannabinoid system, including at a gene level, may have significant implications for the pathogenesis of MOH, and may correlate with psychiatric comorbidities and psychopathological traits.

Supplemental Material

sj-docx-1-cep-10.1177_03331024251314460 - Supplemental material for Genetic characterization of the endocannabinoid system and psychiatric features in patients with migraine and medication overuse headache

Supplemental material, sj-docx-1-cep-10.1177_03331024251314460 for Genetic characterization of the endocannabinoid system and psychiatric features in patients with migraine and medication overuse headache by Marina Romozzi, Lucia Scipioni, Sonia Di Tella, Maria Caterina Silveri, Letizia Maria Cupini, Catello Vollono, Mauro Maccarrone and Paolo Calabresi in Cephalalgia

Footnotes

Acknowledgment

Italian Ministry of Health – Ricerca Corrente 2025.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

Ethical statement

The study adheres to the ethical principles outlined in the 1975 Declaration of Helsinki. It received approval from the Ethics Committee of Università Cattolica del Sacro Cuore in Rome. Informed consent was obtained from qualified patients.

ORCID iDs

Marina Romozzi

Sonia Di Tella

Catello Vollono

Supplemental material

Supplemental material for this article is available online.

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