Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. ...However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CB₁R), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced N-arachidonoylethanolamine (AEA), an endogenous ligand of CB₁R. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.
Abstract
Cannabinoid CB
2
receptor (CB
2
R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the ...discovery of LEI-102, a CB
2
R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB
2
R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB
2
R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB
2
R activation by selective agonists and highlights the role of lipophilicity in CB
2
R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.
Neuroinflammation plays a vital role in Alzheimer's disease and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system, and they play ...essential roles in the maintenance of homeostasis and responses to neuroinflammation. The orphan G-protein-coupled receptor 55 (GPR55) has been reported to modulate inflammation and is expressed in immune cells such as monocytes and microglia. However, its effects on neuroinflammation, mainly on the production of members of the arachidonic acid pathway in activated microglia, have not been elucidated in detail.
In this present study, a series of coumarin derivatives, that exhibit GPR55 antagonism properties, were designed. The effects of these compounds on members of the arachidonic acid cascade were studied in lipopolysaccharide (LPS)-treated primary rat microglia using Western blot, qPCR, and ELISA.
We demonstrate here that the various compounds with GPR55 antagonistic activities significantly inhibited the release of PGE
in primary microglia. The inhibition of LPS-induced PGE
release by the most potent candidate KIT 17 was partially dependent on reduced protein synthesis of mPGES-1 and COX-2. KIT 17 did not affect any key enzyme involved on the endocannabinoid system. We furthermore show that microglia expressed GPR55 and that a synthetic antagonist of the GPR receptor (ML193) demonstrated the same effect of the KIT 17 on the inhibition of PGE
.
Our results suggest that KIT 17 is acting as an inverse agonist on GPR55 independent of the endocannabinoid system. Targeting GPR55 might be a new therapeutic option to treat neurodegenerative diseases with a neuroinflammatory background such as Alzheimer's disease, Parkinson, and multiple sclerosis (MS).
To date,
N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol are the best studied endocannabinoids and are thought to act as retrograde messengers in the central nervous system (CNS). ...By activating presynaptic cannabinoid CB
1 receptors, they can reduce glutamate release in dorsal and ventral striatum (nucleus accumbens) and alter synaptic plasticity, thereby modulating neurotransmission in the basal ganglia and in the mesolimbic reward system. In this review, we will focus on the role of the endocannabinoid system within these neuronal pathways and describe its effect on dopaminergic transmission and vice versa. The endocannabinoid system is unlikely to directly affect dopamine release, but can modify dopamine transmission trough trans-synaptic mechanisms, involving γ-aminobutyric acid (GABA)-ergic and glutamatergic synapses, as well as by converging signal transduction cascades of the cannabinoid and dopamine receptors. The dopamine and endocannabinoid systems exert a mutual control on each other. Cannabinergic signalling may lead to release of dopamine, which can act via dopamine D
1-like receptors as a negative feedback mechanism to counteract the effects of activation of the cannabinoid CB
1 receptor. On the other hand, dopaminergic signalling via dopamine D
2-like receptors may lead to up-regulation of cannabinergic signalling, which is likely to represent a negative feedback on dopaminergic signalling. The consequences of these interactions become evident in pathological conditions in which one of the two systems is likely to be malfunctioning. We will discuss neurological and psychiatric disorders such as Parkinson's and Huntington's disease, drug addiction and schizophrenia. Furthermore, the possible role of the endocannabinoid system in disorders not necessarily depending on the dopaminergic system, such as eating disorders and anxiety, will be described.
Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and ...specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.
Astroglial type‐1 cannabinoid (CB1) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic ...neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB1 receptors. In particular, brain CB1 receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB1 receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock‐out mice lacking astroglial CB1 receptor expression specifically in glial fibrillary acidic protein (GFAP)‐containing astrocytes (GFAP‐CB1‐KO mice) and also generated genetic rescue mice to re‐express CB1 receptors exclusively in astrocytes (GFAP‐CB1‐RS). To better identify astroglial structures by immunoelectron microscopy, global CB1 knock‐out (CB1‐KO) mice and wild‐type (CB1‐WT) littermates were intra‐hippocampally injected with an adeno‐associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP‐CB1‐KO and ‐WT mice, respectively. Furthermore, double immunogold (for CB1) and immunoperoxidase (for GFAP or hrGFP) revealed that CB1 receptors are present in astroglial mitochondria from different hippocampal regions of CB1‐WT, GFAP‐CB1‐RS and GFAPhrGFP‐CB1‐WT mice. Only non‐specific gold particles were detected in mouse hippocampi lacking CB1 receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB1 receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.
Main Points
Intracellular CB1 receptors are localized in astroglial mitochondria.
About 12% of the mitochondria in the hippocampal astrocytes express the receptor.
This novel finding extends the subcellular compartmentalization of the CB1 receptor in the brain.
The endocannabinoid anandamide (
N-arachidonoylethanolamine) was proposed to be an extracellular retrograde messenger, which regulates excitability of neurons by cannabinoid CB
1 receptor-dependent ...inhibition of neurotransmitter release. Recent findings indicate that the neuromodulatory actions of anandamide might be more complex. Anandamide has been shown to directly modulate various ion channels, such as α7-nicotinic acetylcholine receptors, T-type Ca
2+ channels, voltage-gated and background K
+-channels and Transient Receptor Potential Vanilloid type 1 (TRPV1) channels. The binding site of anandamide at some of these ion channels appears to be intracellular or at the bilayer interface. This rises the intriguing possibility that anandamide, prior to its release into the synaptic cleft, may regulate ion homeostasis and excitability of neurons as an intracellular modulator of ion channels independent of its action at cannabinoid CB
1 receptors. This possibility might extend the concept of anandamide as an endocannabinoid retrograde messenger and may have profound implications for its role in neurotransmission and neuronal function. Here, we will review the evidence for this hypothesis.
In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable ...pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2‐cyclopropene‐containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron‐demand Diels–Alder reaction with quenched tetrazine‐fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper‐catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.
This paper describes the use of a cyclopropene‐containing oleic acid analogue for studying live cell lipid uptake by dendritic cells. The minimal oleic acid analogue is incorporated into membranes and lipoproteins, which can be studied by live cell microscopy and lipoproteomics.
Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited ...by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl
-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB
activation. Antinociceptive efficacy of LEI-515 was blocked by CB
, but not CB
, antagonists. The CB
antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.
Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based ...protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and β-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.
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