Endocannabinoid system: key regulatory enzymes and physio-pathological implications
Lipids Signaling

Author: sonila vathi
Date: 05/01/2014


Authors: Sonila Vathi, Simone Schimmenti

The endocannabinoid system consists os several derivatives of arachydonic acid, their receptors and the enzymes involved in their synthesis and degrade such as fatty acid amide hydrolase (FAAH) and monoacyglycerol lipase (MAGL).

Endocannabionids can be isolated from brain and peripheral tissues where they mimic the action of Δ9-tetrahydrocannabinol (THC) in different biological processes.
The main endocannabinoids are:

However the 2 best characterized endocannabionids are Anandamide (AEA) and 2- Arachidonyl glycerol (2-AG) which seem to be the most abundant and those with the higher affinity for the receptors CB1 and CB2.

Endocannabinoids synthesis

Endogenous cannabinoids are produced on-site and on-demand in response to physiological or pathological stimuli. Indeed endocannabinoids, once produced, are not involved in vescicles but they break free from the cell membrane and bind to their receptors in the presinaptic cell. Expression is exclusive, so that both of endocannabinoids are not co-synthesized. This exclusion is based on synthesis-specific channel activation: a recent study found that in the bed nucleus of the stria terminalis, calcium entry through voltage-sensitive calcium channels produced an L-type current resulting in 2-AG production, while activation of mGluR 1/5 receptors triggered the synthesis of anandamide. Ca2+ is need for both mediators synthesis.

Anandamide synthesis

Two biosynthetic pathways have been proposed for AEA synthesis:
1- AEA would be synthesized from free arachydonic acid and ethanolamine. However the reaction is nonspecific and high amounts of reagents are requested to make FAAH (that usaully degradesAEA) in this sense.

2- The second pathway starts with the formation of N- Arachydonil Phosphoethanolamine (NAPE) through the catalysis of a transacylase whose actvity is dependent on Ca2+ presence. The transacylase transferts a unit of arachydonic acid from glycerophopholipid 1-position to the phosphoethanolamine's amine-group. The reaction is selective and involves the acyles in 1 position what limits AEA synthesis and consequently maintains low levels of it in tissues because as it's known arachydonic acid is usually esterified in 2-position. NAPE is then hydrolizated by a D-phospholipase which cleaves the phospholipid in glicerophospholiphid sn-1 releasing the mediator AEA.

NAPE-PLD: a key enzyme in anadamide's synthesis

N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) is currently considered the major enzyme responsible for AEA production.

(Functional Analysis of the Purified Anandamide-generating Phospholipase D as a Member of the Metallo-β-lactamase)

NAPE-PLD is a membrane-bound enzyme, member of the metallo-beta-lactamase family, which hydrolyzes NAPE with extremely high specificity and appears to be constitutively active (membrane components including PE keep the membrane-associated form of NAPE-PLD constitutively active).
The enzyme is activated by increased intracellular Ca2+ concentration. Indeed, millimolar concentrations of Ca2+ are required to cause significant activation of thes enzyme, and several studies have showed that Ca2+ can be replaced by other inorganic divalent cations including Mg2+.

2-AG synthesis

Several biosynthetic routes have been identified in different body tissues suggesting that a process is preffered to another only according to the type of cell that receives the stimulus.

1-The first pathway is thought to produce the endocannabinoid 2-AG starting from inositol phospholipids through 2 different hydrolytic processes: one involves a PLC's action first and then that of a diacyglycerol lipase (DAGL). The other realize 2-AG sythesis through a PLA which forms a lisophosphoinisitol (lisoPI), hydrolized then by a selective lisoIP PLC. Both processes need Ca2+ presence which concentration increases after inositol releasing from cell's membrane promoting 2-AG synthesis.

2-Another way that cells use to produce 2-AG is starting from Phosphataridic acid which is first converted 1-Acyl 2-Arachidonylglicerol and then in 2-AG.

2-AG roles and regulation

It's the most abundant ligand for CB1 and CB2 receptors in the body. Regulates axonal growth and guidance during development, and is required for the generation and migration of new neurons in the adult brain. At developed synapses, 2-AG retrograde signaling inhibits the secretion of both excitatory and inhibitory neurotransmitters controlling neuronal plasticity.

2-AG functions do not involve only cannabinoid receptors. 2-AG is the precursor of arachydonic acid in a pathway that maintains the level of this essential lipid in the brain and other organs. This pathway also drives the cyclooxygenase-dependent generation of inflammatory prostaglandins in the brain, which is implicated in several inflammatory brain pathologies.

(The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling)

Structure: There are two types of DAGL. Both have a 4TM domain that contains a short cytoplasmic N-terminal sequence leading to the 4TM helices, followed by a canonical α/β hydrolase domain that harbours the catalytic activity, followed by a considerable carboxyl-terminal ‘tail’ domain in the case of DAGLα, but not DAGLβ. DAGLα (1042 amino acids) is larger than DAGLβ (672 amino acids).

DAGLα/β make different contributions in 2-AG production within different tissues and DAGLα appears to be much more important in synapse-rich regions. Results from the knockout animals show that they cannot compensate for each other's loss—but nonetheless, there might be some co-operation between the enzymes.

Regulation: The DAGL is positively activated by direct PKA phosphosylation of the catalytic domain, including the regulatory loop. Phosphorylation of the catalytic domain and/or regulatory loop might stabilize an ‘open’ and ‘closed’ conformation. Independent proteomic studies have also shown that both DAGLα and DAGLβ can be palmitoylated, what increases the hydrophobicity of the protein, enhances the membrane association and can serve to modulate a wide range of processes. Furthermore The DAGLa tail mediates the enzyme trafficking and/or retention within specific cellular compartments contributing to a further regulatory process.

An interesting observation is that both DAGLs regulate the number of proliferating neuronal stem cells in the adult brain, and this directly impacts on neurogenesis and its importance in memory, depression and neurodegenerative diseases.

(DAGL-dependent endocannabinoid signalling: roles in axonal pathfinding, synaptic plasticity and adult neurogenesis)

Binding and intracellular effects

Endocannabinoids bind to and activate cannabinoid receptors CB1 and CB2 located in the presinaptic membrane. CB1 and CB2 belong to rodopsin family receptors and share the structure characteristic of all G-protein-coupled receptors: seven transmembrane domains connected by three extracellular and three intracellular loops, an extracellular N-terminal tail, and an intracellular C-terminal tail.

CB1 is predominantly expressed in the CNS: in the brain, cerebellum and spinal cord. CB2 has been mainly localized in peripheral tissues: tonsils, spleen, B and T lymphocytes, in the blood cell, in natural killer (NK) cells.

Endocannabinoids receptors share a complex signal transduction but they essentially have 4 targets: adenylate ciclase, Ca2+ channels, K+ channels, kinases and ceramide.

The modulation of adenylyl cyclase lead to an increase or decrease of cAMP. The increase of cAMP levels is promoted by the binding with a Gi coupled-receptor and reduces the PKA activation. The positive modulation of the enzyme requires the modulator's binding to a heterodimer CB1-D2 that bind to a Gas and activate adenylate cyclase, PKA and CREB.

Endocannabinoids regulate Ca2+ influx through direct and indirect methods: they directly block N, P, Q and L channels as they bind to their receptors. The indirect way is dual and opposing: one pathway is rapid and provide for Ca2+ increase through the By subunit of the G protein, the activation of PLC and IP3 pathway. The second is a long-term pathway due to cAMP inhibition and the consequently RyR phosphorilation by PKA that blocks the release of Ca2 + from intracellular stores. Through the control of calcium endocannabinoids control the neurotramettitors realising from the presynaptic cell and are therefore able to regulate neuronal plasticity.

Regarding the modulation of K + channels endocannabinoids act primarily on channels Kir and Ka bringing to the influx of K+in the presinpatic cell and to its hyperpolarization.

CB1 can be coupled to enzymes that hydrolyze sphingomyelin to ceramide and phosphocholine which act as second messengers. Ceramide activates Raf1 and the ERK cascade through kinases 1/2 as well as factors for the differentiation and proliferation as JNK, and p38 MAPK.

CB1 signalling regulation
CB1 receptors are associated to special membrane microdomains called “lipid rafts” (LR) that modulate CB1-dependent signaling pathways. The functional relationship between CB1and LR is affected by cholesterol content; in particular, membrane cholesterol depletion enhances the binding of CB1 receptors, and subsequent G protein-dependent signaling through AC and MAPK.
Moreover lipid rafts and cholestrol content have an important role in AEA-induced apoptosis. Lipid rafts disruption (or reduced cholesterol content in general) with MCD (methyl-β-cyclodextrin) doubles AEA binding and enhances CB1R signaling leading to a protective effect via increased MAPK activity and decreased release of mitochondrial cytochromec.

(Modulation of the endocannabinoid system by lipid rafts.)

Other EC receptors
CB1 and CB2 receptors are not the only molecular target of endocannabinoids. AEA is able to interact with the orphan receptor GPR55, with the vanilloid receptor TRPV1 and with ionotropic receptors at the peripheral level (ICR). More recently, also nuclear receptors like the peroxisome proliferator-activated receptors (PPARs) have been added to the list of eCBs targets, activated under physiological and pathological conditions.

The TRPV 1 receptor is a non-selective cation channel and its localization on the C fibers makes it important in nociception. The interaction AEA/VR1 occurs in cytosolic space what explains why the inhibition of AMT and FAAH plays a foundamental role in the nociceptive response.

The activation of GPR55, the purported “CB3”, is linked to intracellular Ca2+ increase, activation of the small GTPase proteins RhoA, Rac, and Cdc42 and ERK phosphorylation. Additionally, by triggering PPARs, eCBs exert a variety of long-term effects via genomic mechanisms and rapid non-genomic actions, which are opposite to those evoked by activation of “classical” surface cannabinoid receptors. PPARs activation affects several physiological and pathological processes, such as lipid metabolism, energy balance, and feeding behavior, neuroprotection, epilepsy, circadian rhythms, inflammation, addiction, and cognitive functions.

ECB uptake and degradation

The cannabinoid signal ends as a result of the transport and catabolism of mediators.

The AEA transport into cells depends upon a concentration gradient of unbound anandamide which exists across the cellular membrane. After being transported into the cell, anandamide is subsequently broken down into arachidonic acid and ethanolamine by an enzyme called fatty-acid amide hydrolase (FAAH).
The net movement of anandamide into the cells is coupled to the activity of intracellular FAAH which mantains AEA gradient so that the inhibition of anandamide breakdown with FAAH inhibitors results in its intracellular build-up and the attainment of equilibrium between free intracellular and extracellular anandamide, and this disfavors further net uptake.

(The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase)

FAAH is a serin-hydrolase localized in the membrane of the endoplasmic reticulum of the postsynaptic element. Its catalytic site contains at least two important serines and a lysine with Ser-241 acting as the nucleophile involved in the bond breaking of substrates.

MAGL: the “gate-keeper” enzyme of the endocannabinoid system

2-AG is the most abundant endocannabinoid in the brain and a full agonist of both CB1 and CB2 receptors. It's believed to be hydrolyzed in arachydonic acid and glycerol primarily by the serine hydrolase monoacylglycerol lipase (MAGL) which limits its brain levels and signaling.

(Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system)

Lack of MAGL activity leads to an altered profile of endogenous 2-AG hydrolase activity and dramatic increase of 2-AG levels in the nervous system. Long-term elevation of 2-AG levels leads to desensitization and down regulation of brain CB1 receptors, with a significant reduction of cannabimimetic effects of 2-AG and CB1 agonists proving that MAGL is the major regulator of 2-AG levels and signaling and has a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

In the end both AEA and 2-AG can be transformed in prostaglandins by the COX-2

The DAGL/MAGL pathway is largely responsible for generating and/or maintaining AA levels in the brain and other tissues. An interesting observation is that 2-AG hydrolysis by MAGL generates the prostaglandins that drive inflammatory responses in the brain, including those that lead to neurodegeneration in a mouse model of Parkinson's disease .
As 2-AG is a substrate for COX-2 this might limit eCB signalling in some circumstances, but also generate a novel family of bioactive prostaglandins that act on as yet uncharacterized receptors. Indeed, 2-AG can be oxygenated by a number of lipooxygenases to generate a ligand that can activate peroxisome-activated receptors.

Physio-pathological implications, schizophrenia
Schizophrenia is a mental disease characterized by psychotic episodes with positive symptoms such as hallucinations, delusions and disorganized thinking and speech. Negative symptoms are also present: they commonly include apathy, alogia, anhedonia, asociality, avolition and detachement from reality.
The correlation between this acute disease and the endocannabinoid system can be summarized in these evidences: THC consumption can produce psychotic effects on the subject, as hallucinations and delusions; cannabis gets worse psychotic episodes in schizophrenic patients, as well as increase the incidence in subjects who are prone to develope the illness; arised receptor density has been founded in postmortem examination of patients in anatomic regions involved in schizophrenia; AEA levels are increased in CSF of schizophrenic patients; the treatment with neuroleptics normalizes both endocannabinoid signal and CB1 receptor activity in the nucleus accumbens; hebephrenic schizophrenia is linked with specific polymorphism of CB1.
A gender difference in age of onset of schizophrenia has been consistently reported and a recent meta-analysis has shown that males are on average 1.63 years younger when they first experience psychotic symptoms.

Association of cannabis use with earlier age of onset of schizophrenia
A meta-analysis found that there is a significant increased risk of developing a psychotic disorder in those who have used cannabis. The relationship has been found to be dose dependent and the age of first cannabis use is associated with the age of onset of psychosis.
Earlier age of onset of schizophrenia is generally associated with more severe illness: greater cognitive and functional impairment, more severe symptoms and greater risk of relapse.
Cannabis use may trigger the onset of schizophrenia in individuals who would otherwise have had good prognostic features (i.e.higher premorbid IQ, better functional outcome and less severe symptoms)

(Cannabis use, gender and age of onset of schizophrenia)

High potency cannabis effect and age of first cannabis use related to AOP
The effect of cannabis use on the timing of onset of a psychotic illness is dose dependent.
The association between cannabis use and risk of psychosis is reported to be greater in those who started using cannabis in early adolescence when sensitive brain developmental processes are taking place. Therefore, it is plausible that starting cannabis in early adolescence might also impact on AOP. A younger age at first cannabis use (≤15 years) is associated with a younger AOP only in those who had used cannabis daily. Rather than just measuring the duration of cannabis use, it is important to establish how often and what type of cannabis has been used.

(Daily Use, Especially of High-Potency Cannabis, Drives the Earlier Onset of Psychosis in Cannabis Users)

Correlation between hebephrenic schizophrenia and CNR1 polymorphism
CB1 receptors are encoded by the CNR1 gene. CB1 is located at 6q14 – q15, a site including a schizophrenia susceptibility locus, 6q13 – q26. Two polymorphisms, an AAT-repeat microsatellite in the 3' flanking region and a 1359 G/A polymorphism at codon 453, a silent mutation, in the coding exon of the CNR1 gene, can affect this gene.
The hebephrenic clinical subtype of schizophrenia, but not others, showed a strong association with the CNR1 gene.The genetic evidence indicates that enhanced endocannabinoid activity may be involved in the negative symptoms of schizophrenia.
Enhanced signaling of the cannabinoid system due to heavy consumption of exogenous cannabis or increased endocannabinoid anandamide could precipitate schizophrenia. Increased CB1 receptor density in the prefrontal cortex of schizophrenic patients should facilitate neural transmission of cannabinoids. In addition, genetic variants of the CNR1 gene could alter the efficiency of the receptor function. All of the various findings observed in schizophrenia could converge into hyperactivity of the cannabinoid systems in the brain, which should play an important role in the pathogenesis of schizophrenia.
The recent development of atypical neuroleptics is greatly beneficial to schizophrenic patients because they have been reported to improve not only positive but also negative symptoms with fewer adverse effects than conventional neuroleptics. However, the degree of improvement is not sufficient, especially in negative symptoms. The cannabinoid hypothesis of schizophrenia could be a key to development of aninnovative therapy and comprehensive understanding of the etiology of schizophrenia.

(New Perspectives in the Studies on Endocannabinoid and Cannabis: Cannabinoid Receptors and Schizophrenia)

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