The cannabinoid type 1 receptor (CB1R) was first cloned as the binding receptor for Δ⁹-tetrahydrocannabinol, the main psychologically active compound in Cannabis sativa (14), and CB2R was later cloned in 1993 (15). Since then, a variety of plant-derived and synthetic compounds that target cannabinoid (CB) receptors have been identified and developed as agonists or antagonists. In parallel, endogenous CB ligands were also discovered; anandamide (AEA), which was discovered in 1992 (16), and 2-arachidonoyl glycerol (2-AG), discovered in 1995 (17, 18), are the best-characterized eCB ligands. AEA binds to both CB receptors as a partial agonist, while 2-AG binds to these receptors as a full agonist (19–21). Later on, several new components of the eCB system, including ethanolamine, glycerol, or amino acid derivatives of acyl fatty acids, such as N-palmitoylethanolamine, 2-oleoylglycerol, and N-arachidonoylglycine, were identified in the CNS and shown to be involved in eCB signaling.

CB1R is one of the most abundantly expressed G-protein coupled receptors in the CNS and is primarily expressed in neurons. CB1R is localized in presynaptic terminals where its activation negatively modulates neurotransmission. Thus, CB1R signaling is the critical neuronal regulator for the control of motor function, emotion, cognition, memory, and analgesia (22). CB2R is highly expressed in immune cells, such as B cells, NK cells, and macrophages, in the peripheral nervous system (PNS) and predominantly in microglia in the CNS. Moreover, since CB2R expression is upregulated in tissues under pathological stimuli (23), CB2R is regarded as the central component of the eCB system involving the inflammatory response. With regard to downstream signaling, both CB1R and CB2R have two independent pathways: the canonical G-protein-dependent pathway and the non-canonical G-protein-independent pathway. Upon ligand binding, adenylyl cyclase is inhibited by the activation of G_i/o, the main G protein subunit associated with CBs. As a result, cAMP is reduced, followed by modulation of its downstream signal transducers, such as protein kinase A. CBs are also associated with G_βγ proteins, which initiate other signaling pathways that activate certain calcium and potassium ion channels and several mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinase (ERK), c-Jun NH2 terminal kinase (JNK), and p38 MAPK pathways (24). These pathways are involved in cell proliferation, migration, and cytokine production. Additionally, non-canonical CB signaling can be mediated by β-arrestin (25). β-arrestin is the scaffold protein associated with CBs, and it regulates their internalization and desensitization. β-arrestin and several other signal molecules are recruited to form complexes with CB receptors to act as either receptor signal transducers or terminators (26). Thus, CBs mediate multiple signaling pathways that intricately cross talk with each other. The output signaling is impacted by surrounding microenvironments and intracellular conditions. In addition, the selectivity and preference of downstream CB signaling is determined by the CB ligands, endogenous AEA and 2-AG, or the synthetic CB agonists (27, 28). Considering these CB ligands may also have off-target effects (29), regulation of this complex signaling system by eCB modulation has not yet been completely elucidated.

After the discovery of the endogenous ligands, several enzymes responsible for their biosynthesis and metabolism in the CNS were identified. The major synthesizing enzymes for 2-AG are diacylglycerol lipase (DAGL)α and DAGLβ (30), by which diacylglycerol is converted to 2-AG. DAGLα is the major biosynthesizing enzyme in neurons, while DAGLβ is the major biosynthesizing enzyme in microglia (31). There are multiple pathways responsible for the biosynthesis of AEA; N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), which catalyzes the cleavage of N-acylethanolamine from N-arachidonoyl-phosphatidylethanolamine, is considered the main biosynthetic enzyme (32). 2-AG degradation occurs mainly through monoacylglycerol lipase (MAGL) (33, 34) but also through α-β-hydrolase domain (ABHD)6 (35) and ABHD12 (36) to a lesser extent (37). The chief degrading enzyme of AEA is fatty acid amide hydrolase (FAAH). In addition, eicosanoid biosynthetic enzymes such as cyclooxygeanase-2 (COX-2), lipoxygenase12/15, and cytochrome P450 (CYP450) are also involved in eCB metabolism due to the structural similarity between eCB ligands and the eicosanoid precursor, arachidonic acid (38). In order to boost eCB signaling, several enzyme inhibitors have been developed to block the activity of the eCB-degrading enzymes. The inhibitors of MAGL (i.e., JZL184), ABHD6 (i.e., WWL70), and FAAH (i.e., PF3845, URB597) have been extensively investigated with regard to their pharmacological efficacy in vitro and in in vivo disease models. Figure 1 shows a schematic diagram of CB2R signaling pathways and eCB metabolic pathways, including enzyme inhibitors, in microglia.

As new eCB ligands were discovered, additional receptors were identified and coupled with eCB signaling and function. These receptors include transient receptor potential vanilloid 1 (TRPV1) (39), GPR55 (40), GPR18 (41), and peroxisome proliferator-activated receptors (PPARs) (42). PPARγ is activated when it binds to one of several lipid mediators, such as AEA and other N-acylethanolamines; it then acts as a transcription factor for a subset of genes that are involved in energy and lipid metabolism, oxidative stress inhibition, and the anti-inflammatory response (43). Furthermore, PPARγ has been recognized as a modulator of microglial alternative activation since treatment with a PPARγ agonist triggers alternative activation of microglia in vitro and in a chronic stress model (44).