Receptors and ligands

Shortly after CB₁, a second receptor for cannabinoids was characterized and named cannabinoid receptor 2 (CB₂) (Munro et al. 1993). Both receptors are G-protein coupled, but display distinct expression patterns. CB₁ is most abundant in neuronal tissue (Matsuda et al. 1990) and is detected only at low levels in peripheral organs such as heart, testis and in the immune system (Bouaboula et al. 1993; Galiègue et al. 1995). In contrast, the CB₂ receptor is mainly present in immune cells (Munro et al. 1993; Pacher & Mechoulam 2011). Receptors like the transient receptor potential vanilloid type-1 (TRPV-1) receptor and the orphan G-protein coupled receptor GPR55 are discussed to be non-CB1/CB2

cannabinoid receptors (Zygmunt et al. 1999; Huang et al. 2002; Ryberg et al. 2007). More recently, the orphan G-protein coupled receptor GPR18 was proposed as a possible novel

cannabinoid receptor, as it was shown to be activated by THC and other cannabinoids (McHugh et al. 2012).

Anandamide (arachidonoyl ethanolamine, AEA) and 2-arachidonoyl glycerol (2-AG) were the first endocannabinoids that were characterized and are still considered as the “main endocannabinoids” (Devane et al. 1992; Mechoulam et al. 1995; Sugiura et al. 1995). Both are derived from lipid precursor molecules, similar to the more recently identified endocannabinoids 2-arachidonoyl glycerol ether (noladin ether), 2-arachidonoyl dopamine (NADA) and virodhamine (Hanus et al. 2001; Bisogno et al. 2000; Porter et al.

2002).

1.2.1 Synthesis and degradation of anandamide and 2-AG

Due to their lipophilic nature, anandamide and 2-AG cannot be stored in intracellular vesicles, but have to be produced on demand in an activity-dependent process.

Anandamide is generated from the plasma membrane constituent phosphatidylethanolamine (PE) in a two-step process (see Figure 1 a). First, N-arachidonoyl-phoshatidylethanolamine (NAPE) is formed by N-acylation that is mediated by a membrane bound N-acyltransferase. Subsequently, NAPE is hydrolyzed by a Ca²⁺ -sensitive phosphodiesterase of the phospholipase D type (NAPE-PLD) to N-arachidonoyl ethanolamine (Hansen et al. 2000; Okamoto et al. 2004). The pathway described here is the most direct pathway leading to the generation of anadamide. However, overall four different pathways have been described for its synthesis (for review see: Di Marzo 2008).

2-AG is formed from a diacylglycerol (DAG) molecule that contains arachidonic acid at its sn-2 position (see Figure 1 b). This precursor is provided by phospholipase CPLC)-mediated hydrolysis of the membrane phospholipid phosphatidylinositol (PI). In a second step, the enzyme diacylglycerol lipase (DAGL) hydrolyzes DAG into the monoacyl glycerol 2-AG and fatty acid (Prescotts & Majerus 1983; Sugiura et al. 1995).

Overexpression studies of the DAGL enzymes, pharmacological blockage or knockout approaches indicate that this is the main pathway leading to the generation of 2-AG (Bisogno et al. 2003; Jung et al. 2005; Tanimura et al. 2010). However, other pathways involving the activity of phospholipase-A1 and lyso-PLC have been described (Sugiura et al. 1995).

The degradation of anandamide and 2-AG is mediated by the enzymes fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL), respectively (Figure 1).

FAAH was initially isolated from rat liver membranes and was shown to hydrolyze anandamide into arachidonic acid (AA) and ethanolamine (Cravatt et al. 1996). The enzyme is highly expressed in the central nervous system (CNS), where it is mainly localized at intracellular membranes of Ca²⁺ storing organelles such as mitochondria or

the smooth endoplasmatic reticulum (Gulyas et al. 2004). The serine hydrolase MAGL cleaves 2-AG into arachidonic acid (AA) and glycerol. It was the first enzyme identified to be involved in 2-AG hydrolysis (Dinh et al. 2002). MAGL is abundantly expressed in CB1 -positive nerve terminals and its activity accounts for approximately 80-85 % of total 2-AG degradation in the CNS (Dinh et al. 2002; Dinh et al. 2004; Saario et al. 2005). However, more recently the -hydrolases ABHD6 and ABHD12 have been reported to play a role in the termination of 2-AG signaling. ABHD6 is an integral membrane protein with the active site facing to the cell interior (Blankman et al. 2007). Located on the post-synaptic site of neuronal circuits, ABHD6 monitors the intracellular levels of 2-AG and is responsible for approximately 4 % of 2-AG degradation in the CNS (Blankman et al. 2007;

Marrs et al. 2010). Less is known about ABHD12, which catalyzes up to 9 % of 2-AG inactivation. Like ABHD6, this enzyme is an integral component of the plasma membrane, but its active site is directed towards the extracellular space. In the CNS, this hydrolase was shown to be mainly present on microglia (Fiskerstrand et al. 2010).

Figure 1: Synthesis and degradation of anandamide and 2-AG. a) For the synthesis of anandamide, PE is acylated by N-acyltransferase to produce NAPE, which is subsequently converted to AEA by NAPE-PLD. Degradation of anandamide is mediated by FAAH and results in the generation of AA and ethanolamine. b) The production of 2-AG originates from PI, which is cleaved by PLC to generate DAG. DAG is then further hydrolyzed by DAGL to 2-AG. The enzymes involved in 2-AG degradation are MAGL and ABHD6 and 12. These enzymes convert 2-AG into its constituents AA and glycerol. PE: phosphatidylethanolamine, NAPE: N-arachidonoyl-phosphatidylethanolamine, NAPE-PLD: N-arachidonoyl-phosphatidylethanolamine specific phospholipase D, AEA: anandamide, FAAH: fatty acid amid hydrolase, AA: arachidonic acid, PI:

phosphatidylinositol, PLC: phospholipase C, DAG: diacylglycerol, DAGL: diacylglycerol lipase, 2-AG: 2-arachidonoyl glycerol, MAGL: monoacyl glycerol lipase, ABHD6 /12: -hydrolase 6 and 12

1.2.2 The diacylglycerol lipases (DAGL) and 

DAGL enzymes are responsible for the generation of 2-AG. Two isoforms, termed DAGLand DAGLhave been describedInitially, their enzymatic activity was revealed in human platelets, where stimulation with thrombin led to a DAGL-mediated hydrolysis of DAG (Bell et al. 1980). The first comprehensive characterization of the two isoforms DAGLα and DAGLβ was facilitated by a bio-informatic approach in 2003 (Bisogno et al.

2003). Both enzymes are closely related and display sn-1 specificity regarding to their substrate DAG. Nevertheless, differences were found concerning their expression pattern in the developing organism. While DAGLα is constantly expressed during neuronal development and also in the adult CNS, DAGLβ is less abundant in the adult brain (Bisogno et al. 2003). In contrast, analysis of DAGL and DAGLβ knockout mice revealed that DAGLβ is the main enzyme producing 2-AG in the liver (Gao et al. 2010). During brain development, both enzymes are localized on the axonal tracts and - together with CB₁ - on growth cones of growing neurons, implicating an important function of the ECS in axon guidance and path finding (Bisogno et al. 2003; Berghuis et al. 2007). Later, in the adult brain, DAGLα expression is restricted to the postsynaptic dendritic compartment. As a multi-pass transmembrane protein, it is inserted into the plasma membrane in close proximity to the post-synaptic density (Bisogno et al. 2003). Several reports indicate an important function of DAGL and  not only during brain development, but also during adult neurogenesis in the subventricular zone (SVZ) and the hippocampus (Goncalves et al. 2008; Gao et al. 2010). Thus, the DAGL enzymes are key players of the ECS and their contribution to ECS-mediated functions has to be further investigated.

1.2.3 ECS signaling

The ECS is a complex signaling system that has been shown to be involved in a multitude of physiological processes. Besides its prevalence in the CNS, the ECS exerts important peripheral functions related to immunity, bone metabolism, cardiovascular-, gastrointestinal- and reproductive functioning (Downer 2011; Idris & Ralston 2010;

Montecucco & Di Marzo 2012; Izzo & Sharkey 2010; Battista et al. 2012). However, its subcellular arrangement and operating mode is best characterized in the neuronal environment. Here, the ECS acts as pro-homeostatic, retrograde signaling system in GABAergic- as well as in glutamatergic circuits (for review see: Ohno-Shosaku et al.

2012).

The localization of the individual components of the ECS is representatively depicted for an excitatory glutamatergic nerve terminal in Figure 2. Basal synaptic communication, which is characterized by presynaptic glutamate release upon Ca²⁺ influx

and activation of postsynaptic ionotropic glutamate receptors, does not evoke ECS activity. In contrast, when excessive signaling leads to a massive release of glutamate, postsynaptic type I metabotropic glutamate receptors (mGluR1/mGluR5) become activated and trigger the production of DAG by PLC. Subsequent DAGL activity catalyzes the conversion of DAG into the endocannabinoid 2-AG, which is then released into the synaptic cleft and activates presynaptic CB₁ receptors. Subsequently, CB₁ -associated Gi/o-proteins mediate the inhibition of voltage gated Ca²⁺ channels and reduce neurotransmitter release from the presynaptic site. For the termination of CB₁ activation, 2-AG is rapidly removed from the synaptic cleft and degraded by the presynaptically located MAGL (for review see: Katona & Freund 2008).

Figure 2: Retrograde signaling of the endocannabinoid system exemplarily illustrated for a glutamatergic nerve terminal. a) High frequency stimulation of the presynaptic neuron leads to a Ca²⁺ influx into the presynaptic bouton and subsequently to a massive release of glutamate (Glu) into the synaptic cleft. b) The large amount of glutamate activates postsynaptic AMPA/NMDA receptors, but also metabotropic glutamate receptors (mGluR5), located at the edge of the postsynaptic density. The activated Gq11 protein in turn activates PLCwhich generates DAG from PIP2. DAG is further cleaved by DAGL to 2-AG. 2-AG is released into the synaptic cleft, where it binds and activates presynaptic CB1 receptors. c) The  subunit of the associated Gi protein inhibits voltage gated Ca²⁺ channels, which leads to a reduction of neurotransmitter release from the presynaptic side. d) Rapid uptake of 2-AG and degradation by presynaptic MAGL terminates CB1 signaling. Glu: glutamate, AMPA: -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (ionotropic glutamate receptor), NMDA: N-methyl-D-aspartate receptor (ionotropic glutamate receptor), mGluR5: metabotropic glutamate receptor 5, PLC:

phospholipase-C DAGL: diacylglycerol lipase, PIP2: phosphatidylinositol-2-phosphat, DAG:

diacylglycerol, 2-AG: 2-arachidonoyl glycerol, CB1: cannabinoid receptor 1, MAGL: monoacyl glycerol lipase, red crosses symbolize enhanced vesicle release

As 2-AG and anandamide are hydrophobic messenger molecules. They can easily cross lipid bilayers by diffusion. However, the existence of membrane transporter molecules that mediate the re-uptake these endocannabinoids was proposed by several researchers and is still a matter of debate (for review see: Fowler 2012).

The illustrated mechanism of retrograde signaling ascribes an important role to the ECS in stabilizing the internal environment and supporting the maintenance of healthy conditions (De Petrocellis & Di Marzo 2009). However, ECS signaling is also important under pathological conditions, as for example in neuroinflammatory processes (Centonze et al. 2007). Microglia, the resident immune cells of the CNS, play an important role in coordinating inflammatory reactions (for review see: Graeber et al. 2011). These cells have been shown to communicate via the ECS, thus implicating an important function of this signaling system in the regulation of an inflammatory response (for review see: Stella 2009; Pandey et al. 2009). The following sections will introduce microglia as key players in the immune response of the CNS. In addition, a short overview will be given about how microglia utilize the ECS as an immunomodulatory system.