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In migraine pain, cannabis has a promising analgesic action, which, however, is associated with side psychotropic effects. To overcome these adverse effects of exogenous cannabinoids, we propose migraine pain relief via activation of the endogenous cannabinoid system (ECS) by inhibiting enzymes degrading endocannabinoids. To provide a functional platform for such purpose in the peripheral and central parts of the rat nociceptive system relevant to migraine, we measured by activity-based protein profiling (ABPP) the activity of the main endocannabinoid-hydrolases, mon-oacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). We found that in trigeminal ganglia, the MAGL activity was nine-fold higher than that of FAAH. MAGL activity exceeded FAAH activity also in DRG, spinal cord and brainstem. However, activities of MAGL and FAAH were comparably high in the cerebellum and cerebral cortex implicated in migraine aura. MAGL and FAAH activities were identified and blocked by the selective and potent inhibitors JJKK-048/KML29 and JZP327A, respectively. The high MAGL activity in trigeminal ganglia implicated in the generation of nociceptive signals suggests this part of ECS as a priority target for blocking peripheral mechanisms of migraine pain. In the CNS, both MAGL and FAAH represent potential targets for attenuation of migraine-related enhanced cortical excitability and pain transmission.
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Int. J. Mol. Sci. 2021, 22, 1204. https://doi.org/10.3390/ijms22031204 www.mdpi.com/journal/ijms
Article
Distinct Activity of Endocannabinoid-Hydrolyzing Enzymes
MAGL and FAAH in Key Regions of Peripheral and Central
Nervous System Implicated in Migraine
Adriana Della Pietra 1, Rashid Giniatullin 1,2,* and Juha R. Savinainen 3,*
1 A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70211 Kuopio, Finland;
adriana.della.pietra@uef.fi
2 Laboratory of Neurobiology, Kazan Federal University, Kazan 420008, Russia
3 Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
* Correspondence: rashid.giniatullin@uef.fi (R.G.); juha.savinainen@uef.fi (J.R.S.)
Abstract: In migraine pain, cannabis has a promising analgesic action, which, however, is associated
with side psychotropic effects. To overcome these adverse effects of exogenous cannabinoids, we
propose migraine pain relief via activation of the endogenous cannabinoid system (ECS) by inhib-
iting enzymes degrading endocannabinoids. To provide a functional platform for such purpose in
the peripheral and central parts of the rat nociceptive system relevant to migraine, we measured by
activity-based protein profiling (ABPP) the activity of the main endocannabinoid-hydrolases, mon-
oacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). We found that in trigeminal
ganglia, the MAGL activity was nine-fold higher than that of FAAH. MAGL activity exceeded
FAAH activity also in DRG, spinal cord and brainstem. However, activities of MAGL and FAAH
were comparably high in the cerebellum and cerebral cortex implicated in migraine aura. MAGL
and FAAH activities were identified and blocked by the selective and potent inhibitors JJKK-
048/KML29 and JZP327A, respectively. The high MAGL activity in trigeminal ganglia implicated in
the generation of nociceptive signals suggests this part of ECS as a priority target for blocking pe-
ripheral mechanisms of migraine pain. In the CNS, both MAGL and FAAH represent potential tar-
gets for attenuation of migraine-related enhanced cortical excitability and pain transmission.
Keywords: migraine; pain; endocannabinoid; serine hydrolases; analgesia
1. Introduction
Migraine is a widespread neurovascular disabling disorder affecting up to 15% of
the worldwide population and is typically characterized by one-sided throbbing long-
lasting moderate or severe pain [1,2]. Migraine is associated with multiple psychiatric
comorbidities such as anxiety, depression and panic disorders [3–6]. Despite this clear
association, the CNS neuronal centers underlying the link between migraine and the
comorbid psychiatric conditions remains to be determined. Migraine has a clear trend to
chronicization, namely, a progression from episodic to chronic migraine [7]. This trend
might be due to excessive use of analgesic including opioids drugs leading to condition
known as a ’medication overuse headache’ [8]. Although cannabis can help in opioid de-
toxification [9], its abuse can trigger psychiatric risk factors for migraine such as anxiety
and depression, which are common among cannabis users [10] and associated with the
chronicization of migraine [4,11]. Also, the post-traumatic stress disorder has been shown
to be associated with migraine and drug abuse [12]. The main complaint of migraine pa-
tients is long-lasting pulsating pain, which is intractable in many cases thus leading to
chronic stress and depression [3,13,14]. Known from ancient times [15], cannabinoids
emerged recently as a promising analgesic approach to treat migraine pain [16–18]. In
Citation: Della Pietra, A.;
Giniatullin, R.; Savinainen, J.R.
Distinct Activity of
Endocannabinoid-Hydrolyzing
Enzymes MAGL and FAAH in Key
Regions of Peripheral and Central
Nervous System Implicated in
Migrane. Int. J. Mol. Sci. 2021, 22,
1204. https://doi.org/
10.3390/ijms22031204
Academic Editor: Aron Wellerfigure
Received: 17 December 2020
Accepted: 24 January 2021
Published: 26 January 2021
Publisher’s Note: MDPI stays neu-
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (http://cre-
ativecommons.org/licenses/by/4.0/).
Int. J. Mol. Sci. 2021, 22, 1204 2 of 17
particular, cannabis, now legalized in many countries, has shown a therapeutic effect in
migraine [16,19]. Indeed, marijuana had been used in the past for medicinal purposes to
relieve headaches [20]. However, the exocannabinoids, exogenous compounds from can-
nabis and marijuana, which bind and activate cannabinoid receptors, have many adverse
psychotropic and other unwanted effects [21]. Psychotropicity may be as detrimental as
the migraine condition itself for conducting everyday life. Therefore, an alternative ap-
proach for migraine pain therapy might be based on the selective enhancement of endog-
enous cannabinoids (endoCB), which are naturally generated in the nociceptive system in
the body [22]. The Endocannabinoid System (ECS) is composed of endoCBs, a class of
unique lipidic mediators including 2-arachidonoyl glycerol (2-AG) and anandamide (N-
arachidonoyl ethanolamine, AEA), the metabolic enzymes for their synthesis and degra-
dation along with the two G protein-coupled cannabinoid receptors (CB1 and CB2) [23].
The enhancement of endoCBs-activities is primarily important for conditions such as Clin-
ical Endocannabinoid Deficiency (CECD), which was already proposed as a complication
for several treatment-resistant types of pain, including migraine [16,24].
There are two major types of migraine, migraine with and without aura [25]. Whether
the mechanisms initiating migraine attack are located in the CNS or in the periphery re-
mains debated. The peripheral ‘trigeminovascular system’ (TGVS) is composed by me-
ningeal nerves, vessels and immune cells. In migraine with aura, a plausible early event
is the cortical spreading depolarization (CSD), which leads to global depolarization of
neurons and glial cells [26,27]. The depolarization slowly propagates along the cortical
areas and, leading to activation of the TGVS [26,27]. In the absence of CSD, for instance,
in episodic migraine without aura, the mechanisms triggering migraine are not still clearly
established. However, it has been shown that the potential trigger for migraine attack is
psychogenic stress, which can precipitate or worsen migraine [28,29]. Such stress can pro-
mote the release of corticotropin-releasing hormone [29], directly activating meningeal
mast cells that are closely interacting with the dural nerves in the TGVS [30–32]. Alterna-
tively, stress can provoke sleep disturbances, also known to trigger migraine episodes
[33,34]. Among other key mechanisms initiating migraine attack is the release of the neu-
ropeptides such as calcitonin gene-related peptide (CGRP) and pituitary adenylate
cyclase-activating peptide (PACAP) [35–37]. In most migraine cases, there is an involve-
ment of CNS centers, including the brainstem nuclei and hypothalamus [38].
Taken together, these data suggest that migraine pain may be regulated at multiple
levels, suggesting that pro-nociceptive signaling before or during an attack may be inhib-
ited by endogenous analgesic molecules in the peripheral nervous system (PNS) or within
the CNS.
The analgesic anti-nociceptive potential of cannabinoid CB1 receptors is well estab-
lished [39,40]. Moreover, it has been shown that AEA, one of the key endoCBs, inhibited
trigeminal neurons in animal models of migraine [41,42]. However, the activity profile of
endoCB-degrading enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hy-
drolase (FAAH), targeting 2-AG and AEA respectively, is poorly studied within the noci-
ceptive system. Normally, MAGL and FAAH activity maintains low physiological levels
of endoCBs. High local MAGL and FAAH activity in the PNS and CNS can keep the en-
dogenous analgesic action of endoCBs at low levels, giving rise to CECD [16,24]. Given
that migraine pain is different in the pathogenesis from somatic pain, it needs analgesic
agents specifically targeting TGVS. In this regard, one intriguing issue is whether the pro-
file of MAGL/FAAH activity is different in the trigeminal ganglia (TG) implicated in mi-
graine and dorsal root ganglion (DRG) involved in the transmission of somatic and vis-
ceral pain.
Given the contributing to migraine severity role of psychogenic stress, an additional
promising line of anti-migraine therapy could be the activation of the ECS by selected
plant cannabinoids combined with partner terpenes reducing the level of stress or severity
of comorbid mood disorders [43,44].
Int. J. Mol. Sci. 2021, 22, 1204 3 of 17
Therefore, by proposing MAGL and FAAH as main targets for an innovative multi-
target (analgesic and antidepressant) treatment for migraine, we studied their activity in
the rat PNS and CNS, in areas important for the generation and propagation of migraine-
specific pain signals. The activity of these enzymes was evaluated by a versatile chemo-
proteomic method, activity-based protein profiling (ABPP), utilizing for validation of
specificity the recently developed potent and specific MAGL and FAAH inhibitors [45,46].
2. Results
2.1. Peripheral and Central Activity of the Endocannabinoid-Hydrolyzing Enzymes MAGL and
FAAH
2.1.1. MAGL Activity Prevails at Peripheral Level: Trigeminal Ganglia and Dorsal Root
Ganglia
We found that both in TG and DRG, the basal MAGL activity (treatment with DMSO)
was very high (Figure 1A). In both of these tissues, MAGL activity appeared as two
MAGL-isoforms resulting a double-band. This activity was fully inhibited by the ultrapo-
tent MAGL inhibitor JJKK-048 (100 nM) and almost totally blocked by the specific MAGL
inhibitor KML29 (1 M). A closer analysis of MAGL isoforms revealed that the short
MAGL isoform appeared to be more active than the long isoform in peripheral tissues.
However, both isoforms were equally active in the CNS (Supplementary figure S1). Notice
that, in contrast to MAGL, the basal FAAH activity (selectively inhibited by JZP327A) in
rat TG and DRG was relatively low at these peripheral parts of the nociceptive system
(Figure 1A).
Figure 1B shows that both rat TG and DRG have significantly higher MAGL activity
compared to FAAH. This observation was particularly clear in TG, where the basal MAGL
activity was approximately 9-fold higher compared to that of FAAH. Similarly, MAGL
activity compared to FAAH was 5-fold higher in cervical DRG, 4-fold higher in thoracic
DRG and 11-fold higher in lumbar DRG.
Int. J. Mol. Sci. 2021, 22, 1204 4 of 17
Figure 1. Competitive gel-based activity-based protein profiling (ABPP) reveals higher monoacyl-
glycerol lipase (MAGL) activity over fatty acid amide hydrolase (FAAH) in trigeminal ganglia
(TG) and cervical, thoracic and lumbar dorsal root ganglion (DRG). (A) Rat TG and DRG proteo-
mes were preincubated for 1 h with vehicle (DMSO), the MAGL-inhibitors JJKK-048 (100 nM) and
KML29 (1 M) and FAAH-inhibitor JZP327A (1 M). Then they were labeled with the fluorescent
probe TAMRA-FP, as indicated in Materials and Methods. TAMRA-FP labeled bands (active ser-
ine hydrolases) appear dark after in-gel imaging. FAAH and MAGL were identified based on se-
lective inhibition and their expected molecular weights. Notice that MAGL activity after DMSO
treatment is high whereas the FAAH activity was almost absent. (B) Histograms comparing the
basal activity of MAGL and FAAH in TG and DRG. Basal MAGL activity was approximately 9-
fold higher compared that of FAAH in TG (in a.u., arbitrary units). Similarly, MAGL activity com-
pared to that of FAAH was 5-fold higher in cervical DRG, 4-fold higher in thoracic DRG and
11-fold higher in lumbar DRG. Unpaired t-test, ***p < 0.001, n = 8.
2.1.2. MAGL and FAAH Activity in Brainstem and Spinal Cord
Next, to identify additional molecular targets for analgesia by affecting the most ac-
tive ECS enzymes, we investigated the activity of MAGL and FAAH in central areas in-
volved in the generation and transmission of migraine pain. ABPP testing of rat brainstem
and cervical, thoracic and lumbar spinal cord samples revealed the presence of both
MAGL and FAAH activity (Figure 2A). MAGL activity appeared high in the lumbar
B
A
Trigeminal
Cervical DRG
Lumbar DRG
Thoracic DRG
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
Int. J. Mol. Sci. 2021, 22, 1204 5 of 17
spinal cord, where it was 8-fold higher compared to FAAH activity (Figure 2B). A lower
relative basal MAGL activity against FAAH was observed also in the brainstem (2-fold
higher) and thoracic spinal cord (4-fold higher). In contrast, no significant difference be-
tween basal MAGL and FAAH activities in the cervical spinal cord was found (Figure 2B).
Figure 2. Competitive gel-based ABPP reveals variable MAGL and FAAH activities in rat brain-
stem and cervical, thoracic and lumbar spinal cord. (A) Brainstem and spinal cord proteomes were
incubated for 1 h with vehicle (DMSO), MAGL inhibitors JJKK-048 (100 nM) and KML29 (1 M)
and FAAH inhibitor JZP327A (1 M), and then labeled with the fluorescent probe TAMRA-FP, as
indicated in Materials and Methods. FAAH and MAGL were identified based on selective inhibi-
tion and their expected molecular weights. Note that basal MAGL activity was high in the lumbar
spinal cord but less intense in samples of the brainstem, cervical and thoracic spinal cord. Based
on this analysis, FAAH activity was not found in samples of the brainstem and spinal cord. (B)
Histograms comparing the basal activity of MAGL and FAAH in the brainstem and different spi-
nal cord parts. Basal MAGL activity was approximately 2-fold higher compared to that of FAAH
in brainstem, and 4-fold higher in thoracic spinal cord. In the lumbar spinal cord, MAGL activity
was 8-fold higher than of FAAH. Unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 11 (BS, cSC)
and n = 8 (tSC, lSC).
B
A
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
*
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
ns
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
**
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
Brainstem
Cervical
Spinal Cord
Lumbar
Spinal Cord
Thoracic
Spinal Cord
ßFAAH
ßMAGL
Lumbar
Spinal Cord
Thoracic
Spinal Cord
Cervical
Spinal Cord
Brainstem
DMSO
JJKK-048 (100nM)
KML29 (1uM)
JZP327A (1uM)
DMSO
JJKK-048 (100nM)
KML29 (1uM)
JZP327A (1uM)
DMSO
JJKK-048 (100nM)
KML29 (1uM)
JZP327A (1uM)
DMSO
JJKK-048 (100nM)
KML29 (1uM)
JZP327A (1uM)
35kDa
50kDa
70kDa
100kDa
25kDa
20kDa
Int. J. Mol. Sci. 2021, 22, 1204 6 of 17
2.1.3. MAGL and FAAH Share the Spotlight at Central Cortical Level
Next, we explored whether MAGL and FAAH were active also at the level of CNS.
Using cerebellum and cortex samples (Figure 3A), we observed high MAGL and, for the
first time, relatively high basal FAAH activity in cerebellum, frontal, temporal and occip-
ital cortexes (Figure 3A). Indeed, in the cerebellum, frontal and temporal cortexes, the ba-
sal MAGL activity was only approximately 2-fold higher compared to that of FAAH (Fig-
ure 3B). No significant difference was observed between basal MAGL vs FAAH activities
in the occipital cortex, suggesting a comparable contribution of both of these hydrolases
in control of endoCBs in this important for migraine area of the brain (Figure 3B).
Figure 3. Competitive gel-based ABPP reveals MAGL and FAAH activity in rat cerebellum and cortex. (A) Cerebellar and
frontal, temporal and occipital cortexes proteomes were incubated for 1 h with vehicle (DMSO), MAGL inhibitors JJKK-
048 (100 nM) and KML29 (1 M) and FAAH inhibitor JZP327A (1 M), and then labeled with the fluorescent probe
TAMRA-FP, as indicated in Materials and Methods. FAAH and MAGL were identified based on selective inhibition and
their expected molecular weights. Both MAGL and FAAH activities were high in the cerebellum and cortex. (B) Histo-
grams showing the basal activity of MAGL and FAAH in the cerebellum and frontal, temporal and occipital cortexes.
Basal MAGL activity was 2-fold higher compared to that of FAAH in frontal and temporal cortexes. In contrast, MAGL
and FAAH activities were not found statistically different in samples of the cerebellum and occipital cortex. Unpaired t-
test, *p < 0.05, ***p < 0.001, ns = nonsignificant, n = 10.
B
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
ns
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
***
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
*
MAGL FAAH
0
2
4
6
MAGL activity (a.u.)
ns
Cerebellum Frontal Cortex Occipital Cortex Temporal Cortex
A
Int. J. Mol. Sci. 2021, 22, 1204 7 of 17
2.2. MAGL has a Key Activity at Peripheral Level
Given the overall prevalence of MAGL over FAAH in basal activity in most of the
peripheral and central areas, we also investigated whether MAGL has a similar prevailing
activity in certain areas of the PNS and CNS in comparison with a key migraine-related
tissue such as TG. We found that MAGL activity was higher in peripheral TG than in most
of the other areas (Figures 1A, 2A, 3A and Supplementary Figures S2, S3, S4). In particular,
Figure 4A shows that MAGL activity in TG was 2-fold higher than in thoracic DRG.
However, we could not find a significant difference among activities in TG and cervical
and lumbar DRG (Figure 4A, 1A). Basal MAGL activity in TG prevails on most of the other
tissues: 3-fold higher than in cervical and thoracic spinal cord and 2-fold higher than in
cortexes (Figure 4B, 4C, 1A, 2A, 3A).
Figure 4. Comparing the basal MAGL activity in peripheral and central rat tissues. (A) Comparison of basal MAGL activity
between TG and DRG. Basal MAGL activity was 2-fold higher in TG than in thoracic DRG but no significant difference
was found in TG vs cervical and lumbar DRG. (B) Comparison of basal MAGL activities between peripheral TG and CNS
spinal cord tracts. Basal MAGL activity in the cervical (cervSC) and thoracic (thorSC) spinal cord was 3-fold lower than
in TG. The level of MAGL activity was similar between samples of TG and the lumbar spinal cord (lumbSC). (C) Compar-
ison of basal MAGL activities between peripheral TG and cortical samples. Basal MAGL activity in frontal (FC), temporal
(TC) and occipital cortexes (OC) was approximately half of that in the TG sample. Unpaired t-test, *p < 0.05, **p < 0.01, ns
= nonsignificant, n = 8 (TGs, DRGs), n = 11 (BS, cSC), n = 8 (tSC, lSC) and n = 10 (Cbl, cortex).
2.3. Inhibition of MAGL and FAAH in Peripheral and Central Nervous Tissues
2.3.1. JJKK-048, KML29 and AKU-005 Block Basal MAGL Activity in Both Peripheral and
Central Samples
In order to find the most efficient ways to block MAGL and FAAH activities on TG,
DRG, brainstem, spinal cord and cortex, we numerically evaluated the inhibitory action
on these tissues of the recently proposed MAGL inhibitors JJKK-048 and KML29, FAAH
inhibitor JZP327A and the dual MAGL-FAAH inhibitor AKU-005 (Figure 5, Supplemen-
tary Figures S2, S3, S4). In this testing, we used the fully effective concentrations of the
inhibitors based on our previous studies, determining the dose-responses of these com-
pounds [24–26].
TG cervDRG
0
2
4
6
8
MAGL activity (a.u.)
ns
TG cervSC
0
2
4
6
8
MAGL activity (a.u.)
**
TG FC
0
2
4
6
8
MAGL activity (a.u.)
**
TG thorDRG
0
2
4
6
8
MAGL activity (a.u.)
*
TG thorSC
0
2
4
6
8
MAGL activity (a.u.)
**
TG TC
0
2
4
6
8
MAGL activity (a.u.)
**
TG lumbDRG
0
2
4
6
8
MAGL activity (a.u.)
ns
TG lumbSC
0
2
4
6
8
MAGL activity (a.u.)
ns
TG OC
0
2
4
6
8
MAGL activity (a.u.)
**
A
B
C
Peripheral MAGL basal activity: TG vs DRG
Peripheral TG vs central spinal cord MAGL basal activity
Peripheral TG vs central cortical MAGL basal activity
Int. J. Mol. Sci. 2021, 22, 1204 8 of 17
We found that the specific MAGL inhibitor JJKK-048 (100 nM) almost completely in-
hibited the activity of this ECS enzyme in peripheral parts of the nociceptive system (rep-
resented by TG and DRG). In particular, MAGL activity was reduced by 90% in TG and
cervical DRG, by 85% in thoracic DRG and by 95% in lumbar DRG (Figure 5A). Moreover,
this treatment also inhibited MAGL activity in the CNS. Figure 5B illustrates a reduction
of MAGL activity by 66% in the brainstem (BS), 72% in the cervical spinal cord (cSC), by
68% in the thoracic spinal cord (tSC) and by 79% in the lumbar spinal cord (lSC). JJKK-
048-mediated inhibition at the cortical level was even stronger, with a 95% reduction of
MAGL activity in the frontal cortex (FC) and 90% in temporal and occipital cortexes (TC
and OC, Figure 5C).
Another selective MAGL inhibitor KML29 (1 M) also strongly reduced MAGL ac-
tivity in the PNS, including TG and DRG (by 92% in TG, 88% in cDRG, 86% in tDRG and
93% in lDRG, Figure 5D). Likewise, it also strongly reduced MAGL activity in the cerebral
cortex (by 92% in FC, 88% in TC and 90% in OC; Figure 5F). Moreover, we observed a
noticeable KML29-dependent inhibitory effect on basal MAGL activity on the brainstem
(69%), cervical (69%), thoracic (55%) and lumbar spinal cord (72%) (Figure 5E).
The dual MAGL-FAAH inhibitor AKU-005 (1 M) exhibited the same inhibitory ef-
fect as JJKK-048 and KML29; a strong reduction of MAGL activity was observed at pe-
ripheral (by 93% in TG, 91% in cDRG, 92% in tDRG and 93% in lDRG, Figure 5G) and
cortical level (by 92% in FC, 91% in TC and 90% in OC, Figure 5I). Although, AKU-005
had a moderate inhibitory effect on MAGL activity in the brainstem (77%) and spinal cord
(70% cSC, 78% tSC, we observed a 95% reduction in lSC, Figure 5H).
Figure 5. The blocking capacity of the MAGL inhibitors JJKK-048, KML29 and the dual MAGL-FAAH inhibitor AKU-005
in the CNS and the PNS. (A) JJKK-048 (100 nM) almost completely inhibited MAGL activity at peripheral level: by 90% in
TG and cervical DRG (cDRG), 85% in thoracic DRG (tDRG) and 95% in lumbar DRG (lDRG). (B) JJKK-048 (100 nM) inhib-
ited MAGL activity by 66% in brainstem (BS), 72% in cervical spinal cord (cSC), 68% in thoracic spinal cord (tSC), 79% in
lumbar spinal cord (lSC). (C) JJKK-048 (100 nM) inhibited MAGL activity by 95% in frontal cortex (FC) and 90% in tem-
poral and occipital cortexes (TC and OC). (D) KML29 (1 M) strongly reduced MAGL activity at peripheral level: 92% TG,
88% cDRG, 86% tDRG, 93% lDRG. (E) KML29 (1 M) inhibitory effect on brainstem and cSC was 69%, in tSC 55% and 72%
in lSC. (F) KML29 (1 M) strongly reduced MAGL activity at cortical level: by 92% in FC, 88% in TC, 90% in OC. (G) AKU-
005 (1 M) reduced MAGL basal activity at peripheral level: 93% in TG, 91% in cDRG, 92% in tDRG and 93% in lDRG. (H)
Control TG cDRG tDRG lDRG
0
50
100
MAGL activity (%)
*** *** ***
***
Control TG cDRG tDRG lDRG
0
50
100
MAGL activity (%)
*** *** *** ***
ControlTG cDRG tDRG lDRG
0
50
100
MAGL activity (%)
*** *** *** ***
Control BS cSC tSC lSC
0
50
100
MAGL activity (%)
*** ** ***
***
ControlBS cSC tSC lSC
0
50
100
MAGL activity (%)
*** ***
*
**
Control BS cSC tSC lSC
0
50
100
MAGL activity (%)
*** ***
***
***
Control FC TC OC
0
50
100
MAGL activity (%)
*** *** ***
Control FC TC OC
0
50
100
MAGL activity (%)
*** *** ***
Control FC TC OC
0
50
100
MAGL activity (%)
*** *** ***
A B C
D E F
G H I
JJKK-048 inhibition of MAGL activity
KML29 inhibition of MAGL activity
AKU-005 inhibition of MAGL activity
Int. J. Mol. Sci. 2021, 22, 1204 9 of 17
AKU-005 (1 M) inhibited basal MAGL activity also in brainstem (77%) and spinal cord (70% cSC, 78% tSC, 95% lSC). (I)
AKU-005 (1 M) inhibitory effect on cortical MAGL activity was of 92% in FC, 91% in TC and 90% in OC. One-way
ANOVA with Tukey’s multiple comparison post-hoc test was used for statistical analysis between the MAGL activities
after control (DMSO) and inhibitor treatments, (*p < 0.05, **p < 0.01, ***p < 0.001, ns = nonsignificant). For JJKK-048: n = 8
(TGs, DRGs), n = 11 (BS, cSC), n = 8 (tSC, lSC) and n = 9 (cortex); For KML29: n = 8 (TGs, DRGs), n = 11 (BS, cSC), n = 8 (tSC,
lSC) and n = 9 (cortex); For AKU-005: n = 4 (TGs, DRGs, BS, tSC, lSC), n = 5 (cSC, cortex).
2.3.2. JZP327A Blocks FAAH Activity in the Cerebral Cortex
Unlike the peripheral tissues, as well as the brainstem and spinal cord, we observed
a significantly high basal FAAH activity only in cortical samples (Figure 3). This high ac-
tivity, therefore, represented a reliable model to evaluate specific FAAH inhibitors. In-
deed, the endoCB -hydrolyzing activity of FAAH in the cortex was readily blocked by the
specific FAAH inhibitor JZP327A (1 M) (Figure 6). Thus, JZP327A reduced basal FAAH
activity by 72% in the frontal cortex (FC), 67% in the temporal cortex (TC) and 78% in
occipital cortex (OC) samples.
Figure 6. FAAH inhibitor JZP327A reduces basal FAAH activity in vitro at the cortical level. Data
is from ABPP testing of the frontal cortex (FC), temporal cortex (TC) and occipital cortex (OC) pro-
teomes incubated for 1 h with vehicle (DMSO) and the specific FAAH inhibitor JZP327A (1 M).
Notice that JZP327A reduced FAAH activity by 72% in FC, 67% in TC and 78% in OC. One-way
ANOVA with Tukey’s multiple comparison post-hoc test was used for comparison, ***p < 0.001, n
= 9.
3. Discussion
In this study, we evaluated, for the first time, the activity of the ECS metabolic en-
zymes in the PNS, including the peripheral trigeminovascular nociceptive system, and in
the CNS areas such as the spinal cord, brainstem, cerebellum and cerebral cortex. These
areas are involved in the generation and transmission of migraine pain as well as in other
migraine-related events such as migraine aura. By utilizing a sensitive chemoproteomic
ABPP assay, we profiled the activity of MAGL and FAAH, two major endocannabinoid-
hydrolyzing enzymes in these tissues. Our data suggest MAGL as a potential peripheral
neuronal target for the treatment of migraine pain. At the cortical level, where the activity
of FAAH was similar to MAGL, the dual-inhibition of these enzymatic pathways can at-
tenuate, via raising the levels of two main endoCBs 2-AG and AEA, the phenomenon of
CSD, underlying aura and reduce the central pain transmission. We propose recently de-
veloped potent and selective MAGL and FAAH inhibitors for the activation of ECS in
peripheral and central nervous structures involved in the anti-nociceptive signaling.
3.1. MAGL and FAAH Activity in Peripheral Nervous Systems
By utilizing the ABPP assay, we demonstrated the prevailing active state of the main
endocannabinoid-degrading enzyme MAGL over FAAH in TG, which are the main
Control FC TC OC
0
50
100
FAAH activity (%)
JZP327A inhibition of FAAH activity
***
***
***
Int. J. Mol. Sci. 2021, 22, 1204 10 of 17
constituent of the TGVS, the place where migraine pain originates from [36,47–49]. The
activity of MAGL was higher than the respective activity of FAAH not only in TG, but
also in DRG and brainstem, which are also implicated in the transmission of migraine pain
[50,51]. Interestingly, we observed that the activity of MAGL was higher in TG than in
some DRG suggesting the specific role of this pathway in trigeminal pain, including mi-
graine headache.
One previous study reported high FAAH expression in rat DRG and spinal cord,
suggesting a key role of AEA in modulating peripheral nociceptive signaling [52]. Nota-
bly, our data do not contradict this conclusion as the ABPP assay allowed us to estimate
not only the expression level but also to detect the activity of serine hydrolases in the
peripheral tissues that are implicated in the generation and transmission of migraine pain.
The demonstration of the relatively high peripheral activity of MAGL over FAAH in neu-
ronal tissues, suggests that any increase of 2-AG levels occurring at the periphery during
a migraine attack would be largely damped down by high MAGL activity. This was a
specific reason to suggest the treatment of migraine pain by the MAGL inhibitor to elevate
the level of the anti-nociceptive 2-AG. However, within the TGVS, the generation of pain
involves not only neurons but also vessels and immune cells, in particular, mast cells
[32,53]. Thus, AEA could interfere with these immuno-vascular mechanisms in the me-
ninges, ultimately leading to reduced nociception [54]. This can happen at the level of
meningeal afferents or via the suppressed transmission of peripheral signals to the sec-
ond-order brainstem neurons [16]. Moreover, the role of AEA could be enhanced during
migraine states [55], which are known to be associated with intensive neuro-inflammatory
processes [48]. Therefore, our data do not exclude the role of FAAH/AEA-signalling as a
target for peripheral analgesia but suggests the MAGL/2-AG as the most straightforward
target for anti-nociceptive treatments operating via neuronal mechanisms.
2-AG is the primary endoCB operating via inhibitory Gi/o-protein-coupled CB1 and
CB2 receptors [16]. Indeed, the MAGL-substrate 2-AG was previously found to fully acti-
vate these receptors whereas the FAAH-substrate AEA behaves as a partial agonist at both
receptors [23,56,57]. This view is consistent with our findings on the prevailing activity of
MAGL over FAAH in neuronal tissues at the periphery.
Notably, apart from the accumulation of the analgesic endoCBs 2-AG and AEA, the
inhibition of MAGL and FAAH has additional benefits for anti-nociception, by diminish-
ing the level of endoCB degradation product arachidonic acid which is a precursor for the
pro-inflammatory and pro-nociceptive prostaglandins [16]. Therefore, peripheral MAGL
and FAAH inhibition may have a multicomponent effect on migraine and other types of
inflammatory pain, mediated by mechanical hypersensitivity, and probably, neuropathic
pain, which, like migraine, is characterized by allodynia.
3.2. MAGL and FAAH Activity in the Central Nervous Systems
In contrast to peripheral nociceptive pathways, the activity of FAAH was much
higher in the CNS. However, the activity of MAGL remained high in the brain. The high
activity of these two endoCB-degrading enzymes might indicate a relatively low tonic
inhibitory role of both 2-AG and AEA in modulating central pain processes in healthy
states. In the CNS, the level of 2-AG has some prevalence over AEA [57], suggesting 2-AG
as the primary modulator of synaptic processes in the brain. Nevertheless, both 2-AG and
AEA can serve as the common retrograde messengers released from post-synaptic mem-
branes to target the inhibitory CB1 presynaptic receptors in glutamatergic and GABAergic
synapses [23].
Together, these data indicate a high potential for pharmacological interventions in
the ECS in order to activate, via endoCBs, the inhibitory CB1/2 receptors for the treatment
of migraine pain.
The role of central neuronal networks and brain centers in migraine is region-specific.
Thus, both the brainstem and cervical spinal cord (C1-C3) are implicated in the transmis-
sion of pain signals from the primary afferents to the second-order neurons [17,50,51].
Int. J. Mol. Sci. 2021, 22, 1204 11 of 17
Consistent with this, our study showed that in the cervical spinal cord, unlike other spinal
cord areas, the activity of FAAH was not significantly different from MAGL, reflecting
their specific role in migraine mechanisms.
The occipital cerebral cortex tested in our study, is the common area for the develop-
ment of CSD, a phenomenon underlying aura in the specific form of migraine with aura
[58]. Accordingly, CSD is likely giving rise to multiple visual abnormalities at the initia-
tion of migraine attacks [59–62]. Temporal, frontal cortical lobes and the cerebellum were
also reported to be altered in chronic migraine patients during pain signaling events [63–
66]. In migraine-related cortex and cerebellum, we found the high activity of both MAGL
and FAAH, suggesting a potential reserve for therapeutic interventions against the MAGL
and FAAH activity by their specific inhibitors. Indeed, both MAGL and FAAH signaling
have been shown to modulate pain transmission at central and peripheral levels [67]. It is
generally accepted that CSD is an attractive target for anti-migraine agents [68]. Moreover,
the suppression of CSD by activating CB1 receptors has already been shown [69], implying
that similar effects may be achieved via activation of ECS.
Thus, the enhancement and anti-nociceptive signaling of endoCBs, 2-AG and AEA,
via MAGL and FAAH inhibition, can provide a beneficial reduction of the excessive cor-
tical excitability and attenuate the central pain transmission in migraine and in inflamma-
tory or neuropathic pain.
3.3. Novel Endocannabinoid Hydrolase Inhibitors for the Treatment of Migraine
The identification of the ECS in several CNS areas presents an avenue to pharmaco-
logically enhance the beneficial role of endoCBs in several pathological conditions, includ-
ing pain, cancer, addictive behavior, epilepsy and psychiatric diseases [22,41]. In this
study, we showed for the first time, the comprehensive profile of activity and specific
inhibition of endoCB-hydrolyzing enzymes MAGL and FAAH in tissues of origin and
transmission of migraine pain.
The majority of previously tested MAGL inhibitors lack high selectivity among dif-
ferent hydrolases [70]. In contrast, the recently found KML29 [46] compound has high
MAGL-specificity and has been validated for its analgesic and anti-allodynic effects in
vivo [71–74]. We propose the newly-synthesized highly potent MAGL inhibitor JJKK-048
(IC50 < 0.4 nM) [46,75] as strong prototype drug candidate for migraine analgesia.
Previous studies on FAAH inhibition using OL-135, URB597 [76,77] and PF3845
showed analgesic effects [72,76]. These results raised further interest in the application of
FAAH inhibitors to different pain states and the identification of more efficient and selec-
tive compounds. In our study, we used the recently developed selective FAAH inhibitor
JZP327A [45], which completely blocked FAAH activity in cerebral cortex samples.
An alternative and powerful tool for targeting both MAGL and FAAH in either TGVS
and CNS is the recently developed dual MAGL-FAAH inhibitor AKU-005 that showed a
strong inhibitory effect, even at nanomolar concentrations (IC50 value 0.2–1.1 nM) [46].
Moreover, another dual inhibitor JZL195 has been reported to elicit stronger pain relief
than the other selective MAGL or FAAH inhibitors [78].
3.4. Summary
In summary, we observed distinct profiles of MAGL and FAAH activity in healthy
PNS and CNS. We show that novel selective MAGL and FAAH inhibitors can fully block
the peripheral and cortical activity of these endCB-degrading enzymes in vitro. Our find-
ings highlight MAGL and FAAH as promising targets for novel anti-migraine strategies
via selective enhancement of the anti-nociceptive endoCB drive in this common neurolog-
ical disorder. Future research efforts may focus on testing novel MAGL and FAAH inhib-
itors in in vivo models of migraine.
Int. J. Mol. Sci. 2021, 22, 1204 12 of 17
4. Materials and Methods
4.1. Animals
Animal House of the University of Eastern Finland provided male Wistar rats for this
study. For testing MAGL and FAAH activity, experiments were conducted on organ sam-
ples from 10–12 rats, on occipital cortex slices from 8 rats and trigeminal ganglia fragments
from 7 rats. Animals were housed under the following conditions: 12h dark/light cycle,
grouped housing, ad libitum access to food and water, at an ambient temperature of 22
°C. All experimental procedures performed in this study follow the rules of the European
Community Council Directive of September 22, 2010 (2010/63/EEC). The Animal Care and
Committee of the University of Eastern Finland approved all experimental protocols (li-
cence EKS-008-2019, protocol from 25.11.2019).
4.2. Animals Dissection
4-6 weeks male Wistar rats were dissected according to published protocols to isolate
the trigeminal ganglia [79], cortical areas, brainstem, cerebellum [80] dorsal root ganglia
and spinal cord [81]. The spinal cord was divided into cervical (C2-C8), thoracic (T1–T13)
and lumbar (L1–S4) tracts. We dissected cervical, thoracic and lumbar DRGs following the
same vertebral segmentation.
4.3. Activity-Based Protein Profiling of Serine Hydrolases
Organ samples were mechanically homogenized (glass-glass homogenizer) in ice-
cold PBS, and protein concentrations were determined with BCA protein assay (Pierce,
Rockford, IL, USA), as previously described [82]. Competitive ABPP using tissue homog-
enates was conducted to visualize the selectivity of inhibitors toward endocannabinoid
hydrolases FAAH and MAGL and against other serine hydrolases in tissue proteomes.
We used the active site serine-targeting fluorescent fluorophosphonate probe TAMRA-FP
(ActivX Fluorophosphonate Probes, Thermo Fisher Scientific Inc., Rockford, IL, USA) as
previously described [83]. Briefly, tissue homogenates (100 μg protein) were pre-treated
for 1 h with DMSO or the selected MAGL inhibitors JJKK-048 (School of Pharmacy, UEF)
and KML29 (Cayman Chemicals (Ann Arbor, MI, USA), FAAH inhibitor JZP-327A
(School of Pharmacy, UEF) or the dual MAGL/FAAH inhibitor AKU-005 (School of Phar-
macy, UEF) with indicated concentrations, after which TAMRA-FP incubation was con-
ducted for 1 hour at room temperature (final probe concentration 2 μM) to label active
serine hydrolases. The reaction was quenched by adding 2× gel loading buffer, after which
10 μg protein was loaded per lane and the proteins were resolved in 10 % SDS-PAGE to-
gether with molecular weight standards. TAMRA-FP labelled proteins were visualized by
ChemiDoc™ MP imaging system (BIO-RAD, Hercules, California, USA) with Cy3 blot
application (602/50, Green Epi, Manual Exposure 10s–120s). Quantification of bands was
performed by the software ImageLab (2020 Bio-Rad Laboratories) on the basis of band
intensity (MAGL/FAAH activity, a.u.).
4.4. Statistical Analysis
Data were analyzed using GraphPad Prism 8 (GraphPad Prism Software, La Jolla,
USA). The data are presented as mean SEM (standard error of the mean). Student’s un-
paired t-test and One-way ANOVA with Tukey’s multiple comparison post-hoc test were
used to detect statistical significances.
Supplementary Materials: The following are available online at www.mdpi.com/1422-
0067/22/3/1204/s1, Supplementary materials can be found in the file “Supplementary materials—
Molecular keys for migraine treatment via selective inhibition of endocannabinoid-hydrolyzing en-
zymes”.
Author Contributions: Conceptualization, A.D.P., R.G. and J.R.S.; methodology, A.D.P.; formal
analysis, A.D.P; investigation, A.D.P.; resources, R.G. and J.R.S.; data curation, A.D.P.; writing—
Int. J. Mol. Sci. 2021, 22, 1204 13 of 17
original draft preparation, A.D.P., R.G. and J.R.S.; writing—review and editing, A.D.P., R.G. and
J.R.S.; visualization, A.D.P., R.G. and J.S; supervision, R.G. and J.R.S.; project administration, R.G.
and J.S.; funding acquisition, R.G. and J.R.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the 2019 Migraine Research Foundation Impact Award. RG
and A.D.P were partially supported by the Finnish Academy grant (No 325392) to R.G.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Animal Care and Committee of the University of
Eastern Finland (licence EKS-008-2019 protocol from 25.11.2019).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from
the corresponding author upon reasonable request.
Acknowledgments: We would like to thank Jayendra Z. Patel for providing JZP327A, Taina Vi-
havainen for the help with increasing ABPP assays replicates, Raisa Giniatullina for contributing
to rat samples collection and Sweelin Chew for language editing of the manuscript.
Conflicts of Interest: None of the authors has any conflict of interest to disclose. This paper is in
line with ethical guidelines requested by the journal.
Abbreviations
2-AG 2-arachidonoyl glycerol
ABPP Activity-based protein profiling
AEA N-arachidonoyl ethanol amine, anandamide
BS
CB1, CB2
Cbl
cDRG
CECD
cerv
CGRP
CNS
cSC
CSD
DMSO
DRG
ECS
endoCB
FAAH
FC
lDRG
lSC
lumb
MAGL
NMDA
NMDAR
OC
PACAP
PNS
TC
TG
TGVS
tDRG
thor
tSC
Brainstem
Cannabinoid receptors 1, 2
Cerebellum
Cervical dorsal root ganglia
Clinical endocannabinoid deficiency
Cervical
Calcitonin generelated peptide
Central nervous system
Cervical dorsal root ganglia
Cortical spreading depression
Dimethyl sulfoxide
Dorsal root ganglia
Endocannabinoid system
Endocannabinoids
Fatty acid amide hydrolase
Frontal cortex
Lumbar dorsal root ganglia
Lumbar spinal cord
Lumbar
Monoacylglycerol lipase
N-methyl-D-aspartate
N-methyl-D-aspartate receptors
Occipital cortex
Pituitary adenylate cyclase-activating enzyme
Peripheral nervous system
Temporal cortex
Trigeminal ganglia
Trigeminovascular system
Thoracic dorsal root ganglia
Thoracic
Thoracic spinal cord
Int. J. Mol. Sci. 2021, 22, 1204 14 of 17
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... The endoCBs-degrading enzymes MAGL and FAAH are expressed in structures related to pain origin, nociceptive transmission and perception of pain ( Figure 2B) [46,47]. However, the relative activity of these two enzymes, the major factor determining the functional role of 2-AG and AEA as endogenous analgesics, is not equally present in the PNS and CNS. ...
... However, the relative activity of these two enzymes, the major factor determining the functional role of 2-AG and AEA as endogenous analgesics, is not equally present in the PNS and CNS. As shown in Figure 2B, endoCBs hydrolysis, MAGL and FAAH, are differentially active in the trigeminal ganglion, which is a part of the peripheral nociceptive system and in the brain areas, where pain is finally perceived [47]. Indeed, based on the activity-based protein profiling method (ABPP), identifying active serine hydrolases, including MAGL and FAAH, we found that, in the trigeminal ganglion, the MAGL activity is much higher than that of FAAH ( Figure 2B) [47]. ...
... As shown in Figure 2B, endoCBs hydrolysis, MAGL and FAAH, are differentially active in the trigeminal ganglion, which is a part of the peripheral nociceptive system and in the brain areas, where pain is finally perceived [47]. Indeed, based on the activity-based protein profiling method (ABPP), identifying active serine hydrolases, including MAGL and FAAH, we found that, in the trigeminal ganglion, the MAGL activity is much higher than that of FAAH ( Figure 2B) [47]. Likewise, the level of endoCBs at the periphery is expected to be non-equally present in favor of accumulated AEA, while the amount of 2-AG should be basically low due to the active degradation by MAGL. ...
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Migraine is a disabling neurovascular disorder characterized by severe pain with still limited efficient treatments. Endocannabinoids, the endogenous painkillers, emerged, alternative to plant cannabis, as promising analgesics against migraine pain. In this thematic review, we discuss how inhibition of the main endocannabinoid-degrading enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), could raise the level of endocannabinoids (endoCBs) such as 2-AG and anandamide in order to alleviate migraine pain. We describe here: (i) migraine pain signaling pathways, which could serve as specific targets for antinociception; (ii) a divergent distribution of MAGL and FAAH activities in the key regions of the PNS and CNS implicated in migraine pain signaling; (iii) a complexity of anti-nociceptive effects of endoCBs mediated by cannabinoid receptors and through a direct modulation of ion channels in nociceptive neurons; and (iv) the spectrum of emerging potent MAGL and FAAH inhibitors which efficiently increase endoCBs levels. The specific distribution and homeostasis of endoCBs in the main regions of the nociceptive system and their generation ‘on demand’, along with recent availability of MAGL and FAAH inhibitors suggest new perspectives for endoCBs-mediated analgesia in migraine pain.
... Interestingly, 2-AG was less effective in our experiments. despite the fact that it is present in the brain in higher concentrations than AEA and can stimulate equally through CB1Rs and CB2Rs [68]. It may potentially be due to lower levels of 2-AG access to the presynaptic CB1Rs, as well as to the fact that the 2-AG-degrading enzyme is closer to the CB1R target than AEA. ...
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The endocannabinoid system (ECS) plays an important role in pain processing and modulation. Since the specific effects of endocannabinoids within the orofacial area are largely unknown, we aimed to determine whether an increase in the endocannabinoid concentration in the cerebrospinal fluid (CSF) caused by the peripheral administration of the FAAH inhibitor URB597 and tooth pulp stimulation would affect the transmission of impulses between the sensory and motor centers localized in the vicinity of the third and fourth cerebral ventricles. The study objectives were evaluated on rats using a method that allowed the recording of the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation and URB597 treatment. The amplitude of ETJ was a measure of the effect of endocannabinoids on the neural structures. The concentrations of the endocannabinoids tested (AEA and 2-AG) were determined in the CSF, along with the expression of the cannabinoid receptors (CB1 and CB2) in the tissues of the mesencephalon, thalamus, and hypothalamus. We demonstrated that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was significantly increased in the CSF after treatment with a FAAH inhibitor, while tooth pulp stimulation had no effect on the AEA and 2-AG concentrations in the CSF. We also found positive correlations between the CSF AEA concentration and cannabinoid receptor type 1 (CB1R) expression in the brain, and between 2-AG and cannabinoid receptor type 2 (CB2R), and negative correlations between the CSF concentration of AEA and brain CB2R expression, and between 2-AG and CB1R. Our study shows that endogenous AEA, which diffuses through the cerebroventricular ependyma into CSF and exerts a modulatory effect mediated by CB1Rs, alters the properties of neurons in the trigeminal sensory nuclei, interneurons, and motoneurons of the hypoglossal nerve. In addition, our findings may be consistent with the emerging concept that AEA and 2-AG have different regulatory mechanisms because they are involved differently in orofacial pain. We also suggest that FAAH inhibition may offer a therapeutic approach to the treatment of orofacial pain.
... Alterations in the ECS have been reported in experimental models of various brain disorders, including Alzheimer's disease, Parkinson's disease, and migraine [4][5][6][7]. To certain extent, the reported findings were supported by those obtained in clinical studies [8][9][10]. ...
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The endocannabinoid system (ECS) is implicated in various brain disorders. Changes in the composition of the cerebrospinal fluid (CSF) may be associated with ECS-related pathologies. Endocannabinoids (eCBs) and their analogues are present at low concentrations in human CSF, which hampered the investigation of the ECS in this body fluid. In this study, we developed a highly sensitive and selective micro-flow liquid chromatography-tandem mass spectrometry (micro-LC-MS/MS) method for the analysis of eCBs and eCB analogues in human CSF. The developed method allowed for the quantitative analysis of 16 eCBs and their analogues in human CSF. Micro-LC-MS/MS analyses were performed at a flow-rate of 4 μL min⁻¹ with a 0.3-mm inner diameter column. A minor modification of a novel spray needle was carried out to improve the robustness of our method. By using an injection volume of 3 μL, our method reached limits of detection in the range from 0.6 to 1293.4 pM and limits of quantification in range from 2.0 to 4311.3 pM while intra- and interday precisions were below 13.7%. The developed workflow was successfully used for the determination of eCBs in 288 human CSF samples. It is anticipated that the proposed approach will contribute to a deeper understanding of the role of ECS in various brain disorders.
... Inhibition of FAAH activity by URB597 resulted in an increase in AEA and CB1 receptor levels, as well as a decrease in CB2 receptor expression. Interestingly, 2-AG was less effective in our experiments despite the fact that it is present in higher concentrations in the brain than AEA and can induce stimulation equally through CB1 and CB2 receptors [58]. It may be due to lower levels of 2-AG access to the presynaptic CB1 receptors, as well as to the fact that the 2-AG-degrading enzyme is closer to the target of the CB1R than AEA. ...
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Endocannabinoids act as analgesic agents in a number of headache models. However, their effectiveness varies with the route of administration and the type of pain. In this study, we assessed the role of the fatty acid amide hydrolase inhibitor URB597 in an animal model of orofacial pain based on tooth pulp stimulation. More specifically, we assessed the effects of intracerbroventricular (i.c.v.) and intraperitoneal (i.p.) administration of URB597 on the amplitude of evoked tongue jerks (ETJ) in rats. The levels of the investigated mediators anandamide (AEA), 2-arachidonyl glycerol (2-AG), Substance P (SP), calcitonin-gene-related peptide (CGRP), endomorphin-2 (EM-2) and fatty acid amide hydrolase (FAAH) inhibitor by URB597 and receptors cannabinoid type-1 receptors (CB1R), cannabinoid type-2 receptors (CB2R) and µ-opioid receptors (MOR) were determined in the mesencephalon, thalamus and hypothalamus tissues. We have shown that increasing endocannabinoid AEA levels by both central and peripheral inhibition of FAAH inhibitor by URB597 has an antinociceptive effect on the trigemino-hypoglossal reflex mediated by CB1R and influences the activation of the brain areas studied. On the other hand, URB597 had no effect on the concentration of 2-AG in the examined brain structures and caused a significant decrease in CB2R mRNA expression in the hypothalamus only. Tooth pulp stimulation caused in a significant increase in SP, CGRP and EM-2 gene expression in the midbrain, thalamus and hypothalamus. In contrast, URB597 administered peripherally one hour before stimulation decreased the mRNA level of these endogenous neuropeptides in comparison with the control and stimulation in all examined brain structures. Our results show that centrally and peripherally administered URB597 is effective at preventing orofacial pain by inhibiting AEA catabolism and reducing the level of CGRP, SP and EM-2 gene expression and that AEA and 2-AG have different species and model-specific regulatory mechanisms. The data presented in this study may represent a new promising therapeutic target in the treatment of orofacial pain.
... A certain limitation of our study is that it is a narrative review and not a systematic one, but considering the aforementioned limits of the studies found (the small sample sizes, the lack of placebo-controlled studies, the often-retrospective design, the different titrations of cannabinoid preparations and the different routes of administration), the results of a systematic review would not have been too dissimilar from a narrative one. The adverse events linked to the modulation of the ECS, increasing eCBs, are still uncertain and should be properly assessed, because, although some authors believe it may be a relatively safe option [59], a recent clinical trial with a FAAH inhibitor (in this case, not used for migraine) was interrupted as a result of serious adverse events [60]. In conclusion, promising data are emerging on the possible role of ECS in migraine. ...
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Full-text available
The endocannabinoid system (ECS) influences many biological functions, and hence, its pharmacological modulation may be useful for several disorders, such as migraine. Preclinical studies have demonstrated that the ECS is involved in the modulation of trigeminal excitability. Additionally, clinical data have suggested that an endocannabinoid deficiency is associated with migraine. Given these data, phytocannabinoids, as well as synthetic cannabinoids, have been tried as migraine treatments. In this narrative review, the current clinical evidence of potential ECS involvement in migraine pathogenesis is summarized. Furthermore, studies exploring the clinical effects of phytocannabinoids and synthetic cannabinoids on migraine patients are reviewed.
... Increasing knowledge of the pathophysiology of migraine has allowed new research into disease treatment options that may modulate the underlying trigeminocervical complex activation. In this context, distinct profiles of MAGL and FAAH inhibitors in the PNS and CNS have recently been reported.83,85,96 In recent years, we have also shown that FAAH and MAGL inhibitor effects at the peripheral and central levels are involved in the modulation of migraine-related pain. ...
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Background: Migraine is a complex and highly disabling neurological disease whose treatment remains challenging in many patients, even after the recent advent of the first specific-preventive drugs, namely monoclonal antibodies that target calcitonin gene-related peptide. For this reason, headache researchers are actively searching for new therapeutic targets. Cannabis has been proposed for migraine treatment, but controlled clinical studies are lacking. A major advance in cannabinoid research has been the discovery of the endocannabinoid system (ECS), which consists of receptors CB1 and CB2; their endogenous ligands, such as N-arachidonoylethanolamine; and the enzymes that catalyze endocannabinoid biosynthesis or degradation. Preclinical and clinical findings suggest a possible role for endocannabinoids and related lipids, such as palmitoylethanolamide (PEA), in migraine-related pain treatment. In animal models of migraine-related pain, endocannabinoid tone modulation via inhibition of endocannabinoid-catabolizing enzymes has been a particular focus of research. Methods: To conduct a narrative review of available data on the possible effects of cannabis, endocannabinoids, and other lipids in migraine-related pain, relevant key words were used to search the PubMed/MEDLINE database for basic and clinical studies. Results: Endocannabinoids and PEA seem to reduce trigeminal nociception by interacting with many pathways associated with migraine, suggesting a potential synergistic or similar effect. Conclusions: Modulation of the metabolic pathways of the ECS may be a basis for new migraine treatments. The multiplicity of options and the wealth of data already obtained in animal models underscore the importance of further advancing research in this area. Multiple molecules related to the ECS or to allosteric modulation of CB1 receptors have emerged as potential therapeutic targets in migraine-related pain. The complexity of the ECS calls for accurate biochemical and pharmacological characterization of any new compounds undergoing testing and development.
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Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are promising targets for neuropathic pain and other CNS disorders. Based on our previous lead compound SIH 3, we designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones and evaluated for FAAH and MAGL inhibition properties. Most of the compounds showed potency towards both enzymes with leading FAAH selectivity. Compound (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC50 = 11 nM) and MAGL (IC50 = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Molecular docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π–π stacking via phenyl ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by molecular simulation studies. PAMPA assay revealed that the lead compound is suitable for blood-brain penetration. The lead compound showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo experiments unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compound exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compound can be a potential template for the development of agents for neuropathic pain.
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Serotonin is traditionally considered as a key mediator implicated in migraine. Multiple 5‐HT receptor subtypes contribute to a variety of region‐specific functional effects. The raphe nuclei control nociceptive inputs by releasing serotonin in the brainstem, whereas dural mast cells provide the humoral source of serotonin in the meninges. Triptans (5‐HT1B/D agonists) and ditans (5‐HT1F agonists) are the best‐established serotonergic anti‐migraine agents. However, activation of meningeal afferents via ionotropic 5‐HT3 receptors results in long‐lasting excitatory drive suggesting a pro‐nociceptive role for these receptors in migraine. Nevertheless, clinical data do not clearly support the applicability of currently available 5‐HT3 antagonists to migraine treatment. The reasons for this might be the presence of 5‐HT3 receptors on inhibitory interneurons dampening the excitatory drive, a lack of 5‐HT3A‐E subunit‐specific antagonists and gender/age‐dependent effects. This review is focusing on the controversial role of 5‐HT3 receptors in migraine pathology and related pharmacological perspectives of serotonergic ligands.
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Migraine patients often report (inter)ictal hypersensitivity to light, but the underlying mechanisms remain an enigma. Both hypo-and hyperresponsivity of the visual network have been reported, which may reflect either intra-individual dynamics of the network or large inter-individual variation in the measurement of human visual evoked potential data. Therefore, we studied visual system responsivity in freely behaving mice using combined epidural electroencephalography and intracortical multi-unit activity to reduce variation in recordings and gain insight into visual cortex dynamics. For better clinical translation, we investigated transgenic mice that carry the human pathogenic R192Q missense mutation in the α 1A subunit of voltage -gated Ca V 2.1 Ca 2+ channels leading to enhanced neurotransmission and familial hemiplegic migraine type 1 in patients. Visual evoked potentials were studied in response to visual stimulation paradigms with flashes of light. Following intensity-dependent visual stimulation, FHM1 mutant mice displayed faster visual evoked potential responses, with lower initial amplitude, followed by less pronounced neuronal suppression compared to wild-type mice. Similar to what was reported for migraine patients, frequency-dependent stimulation in mutant mice revealed enhanced photic drive in the EEG beta-gamma band. The frequency-dependent increases in visual network responses in mutant mice may reflect the context-dependent enhancement of visual cortex excitability, which could contribute to our understanding of sensory hypersensitivity in migraine. K E Y W O R D S Ca V 2.1 calcium channels, electrophysiology, sensory systems, visual stimulation
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Background Cortical spreading depression (CSD) underlies the neurobiology of migraine with aura (MWA). Animal studies reveal networks of microvessels linking brain‐meninges‐bone marrow. CSD activates the trigeminovascular system, evoking a meningeal inflammatory response. Accordingly, this study examines the upregulation of an inflammatory marker in extra‐axial tissues in migraine with visual aura. Methods We used simultaneously acquired ¹¹C‐PBR28 PET/MRI data of 18 kDa translocator protein (an inflammatory marker) in MWA patients (n = 11) who experienced headaches and visual aura in the preceding month. We measured mean tracer uptake (SUVR) in four regions of interest comprising the meninges plus the adjacent overlying skull bone (parameningeal tissues, PMT). These data were compared to healthy controls and patients with pain (chronic low‐back pain, CLBP). Results MWA had significantly higher mean SUVR in PMT overlying occipital cortex than both other groups, though not in the PMT overlying three other cortical areas. A positive correlation was also found between the number of visual auras and tracer uptake in occipital PMT. Interpretation A strong persistent extra‐axial inflammatory signal was found in meninges and calvarial bone overlying the occipital lobe in migraine with visual auras. Our findings are reminiscent of CSD‐induced meningeal inflammation and provide the first imaging evidence implicating inflammation in the pathophysiology of migraine meningeal symptoms. We suspect that this inflammatory focus results from a signal that migrates from underlying brain and if so, may implicate newly discovered bridging vessels that crosstalk between brain and skull marrow, a finding of potential relevance to migraine plus other neuroinflammatory brain disorders. This article is protected by copyright. All rights reserved.
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Premise Headaches are a serious public health concern of our days, affecting about 50% of the world’s adult population. However, such a plague is not limited to the modern era, since ancient archaeological, written, religious and cultural evidences testify to countless attempts to face such disorders from medical, neurosurgical, psychological and sociological perspectives. Background Substantially, the Hippocratic and Galenic theories about headache physiopathology remained predominant up to the 17th century, when the vascular theory of migraine was introduced by Thomas Willis and then evolved into the actual neurovascular hypothesis. The medieval Medical School of Salerno, in southern Italy, where the Greco-Roman medical doctrine was deeply affected by the medio-oriental influence, gave particular attention to both prevention and treatment of headaches. Conclusion The texts of the School, a milestone in the literature of medicine, translated into different languages and widespread throughout Europe for centuries, provide numerous useful recipes and ingredients with an actually proven pharmacological efficacy.
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Background: Migraine is a common disabling neurological disorder where attacks have been recognized to consist of more than headache. The premonitory, headache, and postdromal phases are the various phases of the migraine cycle, where aura can occur before, during, or after the onset of pain. Migraine is also associated with photosensitivity and cranial autonomic symptoms, which includes lacrimation, conjunctival injection, periorbital edema, ptosis, nasal congestion, and rhinorrhoea. This review will present the current understanding of migraine pathophysiology and the relationship to the observed symptoms. Evidence acquisition: The literature was reviewed with specific focus on clinical, neurophysiological, functional imaging, and preclinical studies in migraine including the studies on the role of calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP). Results: The phases of the migraine cycle have been delineated by several studies. The observations of clinical symptoms help develop hypotheses of the key structures involved and the biochemical and neuronal pathways through which the effects are mediated. Preclinical studies and functional imaging studies have provided evidence for the role of multiple cortical areas, the diencephalon, especially the hypothalamus, and certain brainstem nuclei in the modulation of nociceptive processing, symptoms of the premonitory phase, aura, and photophobia. CGRP and PACAP have been found to be involved in nociceptive modulation and through exploration of CGRP mechanisms, new successful treatments have been developed. Conclusions: Migraine is a complex neural disorder and is important to understand when seeing patients who present to neuro-ophthalmology, especially with the successful translation from preclinical and clinical research leading to successful advances in migraine management.
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