Dissecting the Signaling Pathways Involved in the Crosstalk Between

Dissecting the signaling pathways involved in the

crosstalk between mGlu5 and CB1 receptors

Downloaded from

Isabella G. Olmo, Talita H. Ferreira-Vieira and Fabiola M. Ribeiro

molpharm.aspetjournals.org

Department of Biochemistry and Immunology, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 31270-901: IGO, THV and FMR. at ASPET Journals on September 26, 2021

Running title page

Running title: mGlu5/CB1 cell signaling crosstalk

Corresponding author: Dr. Fabiola M. Ribeiro, Departamento de Bioquimica e

Imunologia, ICB, Universidade Federal de Minas Gerais, Ave. Pres. Antonio Carlos

6627, Belo Horizonte, MG, Brazil, CEP: 31270-901, Tel.: 55-31-3409-2655; Downloaded from [email protected]

Co-corresponding author: Dr. Talita H. Ferreira-Vieira, Departamento de Bioquimica e molpharm.aspetjournals.org Imunologia, ICB, Universidade Federal de Minas Gerais, Ave. Pres. Antonio Carlos

6627, Belo Horizonte, MG, Brazil, CEP: 31270-901, Tel.: 55-31-3409-3022; [email protected] at ASPET Journals on September 26, 2021 Additional informations:

Number of text pages - 17

Number of tables – 2

Number of figures – 3

Number of references – 164

Number of words in abstract - 110

Number of words in the text - 4161

Standard Abbreviations 2-AG 2-arachidonoyl glycerol AEA arachidonoyl ethanolamide or anandamide AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BDNF Brain-derived neurotrophic factor cAMP cyclic adenosine monophosphate CB cannabinoid CDPPB 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide CHPG (RS)-2-chloro-5-hydroxyphenylglycine CNS central nervous system CREB cAMP response element-binding protein

2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4- Downloaded from CTEP yl)ethynyl)pyridine DAG diacylglycerol DGL diacylglycerol lipase DHPG 3,5-dihydroxyphenylglycine

Elk-1 ETS domain-containing protein molpharm.aspetjournals.org ERK1/2 extracellular signal-regulated kinases FAAH fatty acid amide hydrolase FMR1 fragile X mental retardation 1 gene FMRP fragile X mental retardation protein FXS Fragile X syndrome GPCR G-protein-coupled receptors at ASPET Journals on September 26, 2021 iGluRs ionotropic glutamate receptor

IP3 inositol 1,4,5-triphosphate

IP3R inositol 1,4,5-triphosphate receptor JZL-184 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate LTD long term depression LTP long term potentiation MAGL monoacylglycerol lipase MAPK mitogen-activated protein kinase mGlu metabotropic glutamate MPEP 2-Methyl-6-(phenylethynyl)-pyridine MTEP 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-pyridine NAM negative allosteric modulator NMDA N-methyl-D-aspartate PAM positive allosteric modulators PDK1 pyruvate dehydrogenase kinase 1 PI3K phosphoinositide 3-kinase PIKE phosphoinositide 3-kinase enhancer PIP3 Phosphatidylinositol (3,4,5)-trisphosphate PKC protein kinase C PLCβ phospholipase Cβ PSD postsynaptic density proteins

URB597 Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (R)-(+)-[2,3-dihydro-5-methyl-3-4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin- WIN 55,212 6-yl]-1-naphthalenylmethanone mesylate

Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Abstract

The metabotropic glutamate (mGlu) receptor 5 and the cannabinoid type 1 (CB1) receptor are G-protein-coupled receptors (GPCR) that are widely expressed in the central nervous system (CNS). mGlu5 receptors, present at the postsynaptic site, are coupled to Gαq/11 proteins and display an excitatory response upon activation, while the

CB1 receptor, mainly present at presynaptic terminals, is coupled to the Gi/o protein and Downloaded from triggers an inhibitory response. Recent studies suggest that the glutamatergic and endocannabinoid systems exhibit a functional interaction to modulate several neural

processes. In this review we discuss possible mechanisms involved in this crosstalk molpharm.aspetjournals.org and its relationship with physiological and pathological conditions, including nociception, addiction and fragil X syndrome. at ASPET Journals on September 26, 2021

The glutamatergic system and the mGlu5 receptor

Glutamate, the most important excitatory neurotransmitter in the CNS of mammals, modulates several neural events, including development and cognition, as well as neurological pathogenesis. Glutamate exerts its actions by binding and activating glutamatergic receptors, which are classified into two distinct groups: ionotropic (iGluRs) and metabotropic (mGluRs) receptors. The iGluRs are ligand-gated ion channels and are subdivided into three types: N-methyl-D-aspartate (NMDA), α- Downloaded from amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors

(Karakas et al., 2015). mGluRs are G-protein coupled receptors (GPCRs) that can

trigger cell signaling transduction pathways through intracellular second messengers to molpharm.aspetjournals.org modulate synaptic activity. Eight different mGluRs are subdivided into three groups (I,

II, and III) based on signal transduction, sequence homology, and pharmacological properties (Nakanishi, 1992). Group I includes the mGlu1 and mGlu5 receptors, which at ASPET Journals on September 26, 2021 mediate excitatory responses through the activation of phosphoinositide signaling pathways, and group II and III receptors, which inhibit the cyclic adenosine monophosphate (cAMP) signaling pathway (Nakanishi, 1992).

mGlu5 receptor: Pharmacology and cell signaling pathways

Several studies indicate that the mGlu5 receptor is central in various brain processes, including long term potentiation (LTP) and long term depression (LTD), mechanims important for memory acquisition and learning (Bliss and Collingridge,

1993; Hou and Klann, 2004; Hullinger et al., 2015; Sung et al., 2001). Moreover, relevant neuronal processes such as cell differentiation, plastic alterations, and cell death are also modulated by mGluRs (Erichsen et al., 2015; Sheng and Kim, 2002).

mGlu5 receptors exhibits a three dimensional structure commonly observed among mGluR family members, including a large extracellular amino-terminal portion where the orthosteric site is located, a transmembrane heptahelical domain that contains an allosteric binding site, and an intracellular C-terminal portion (Nakanishi and Masu,

1994). The mGlu5 receptor is broadly expressed in the CNS and is predominantly located perisynaptically near postsynaptic elements and in glial cells (Cai et al., 2000 ;

Lujan et al., 1996; Nusser et al., 1994; Shigemoto et al., 1993). Notably, the mGlu5 Downloaded from receptor is expressed in different brain regions, including the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, cerebral cortex, hippocampus, septum side,

striatum, nucleus accumbens, inferior colliculus, and spinal trigeminal nuclei (Abe et molpharm.aspetjournals.org al., 1992).

mGlu5 receptors can activate multiple cell signaling pathways (Fig. 1). The classical canonical form of the mGlu5 receptor signaling starts with glutamate binding at ASPET Journals on September 26, 2021 to the orthosteric site of the receptor. The ligand/receptor interaction induces Gαq/11 protein activation, which leads to the activation of phospholipase Cβ (PLCβ) and the formation of second messengers, including diacylglycerol (DAG) and inositol 1,4,5- trisphosphate (IP3). IP3 binds to its receptor in the endoplasmic reticulum (IP3R), and this leads to an increase in intracellular Ca2+ levels and consequent activation of protein kinase C (PKC) (Berridge and Irvine, 1984). Simultaneously, PKC activation drives mGlu5 receptor desensitization through phosphorylation (Alaluf et al., 1995; Dhami and

Ferguson, 2006; Ribeiro et al., 2009; Ribeiro et al., 2010).

mGlu5 receptor activation can facilitate its interaction with postsynaptic proteins, triggering the activation of other cell signaling cascades (Tu et al., 1999). Homer-type proteins are mainly postsynaptic, acting as scaffold proteins and binding directly to the carboxy-terminal portion of mGlu5 receptor (Brakeman et al., 1997). Notably, Homer

Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. MOL #104372 proteins appear to be involved in the anchorage and distribution of mGluR5 at the periphery of the synapse and in the expression of mGlu5 receptors at the plama membrane (Ango et al., 2002; Mansouri et al., 2015; Roche et al., 1999). Moreover, mGlu5/homer interaction may also interfere with the regulation of synaptic transmission, as it has been reported that NMDAR activity is controlled by mGlu5 receptor through the protein complex formed by Homer (Moutin et al., 2012).

Moreover, Homer proteins may control spontaneous activity of group 1 mGlu receptors Downloaded from (Ango et al., 2001). Homer proteins are widely expressed in neurons and form complexes with postsynaptic density proteins (PSD) to facilitate the interaction between

plasma membrane receptors and intracellular signal transduction components (Shiraishi- molpharm.aspetjournals.org

Yamaguchi and Furuichi, 2007; Xiao et al., 1998). For example, Homer binding to

IP3Rs brings mGlu5 receptor into close proximity, facilitating the activation of IP3Rs

2+ and the release of Ca from intracellular stores (Tu et al., 1998). mGlu5 receptor at ASPET Journals on September 26, 2021 activation can trigger phosphorylation of extracellular signal-regulated kinases

2+ (ERK1/2) and part of this effect is due to signaling mediated by IP3/Ca (Jong et al.,

2009; Mao et al., 2005; Wang et al., 2007). However, it has been shown that Homer establishes an alternative signaling pathway linking mGlu5 receptor to ERK1/2 in a

2+ 2+ Ca -independent manner (Fig. 1) (Mao et al., 2005). Moreover, both IP3/Ca - and

Homer-dependent cascades are necessary to activate ERK1/2 to levels high enough to phosphorylate the transcription factors ETS domain-containing protein (Elk-1) and cAMP response element-binding protein (CREB), facilitating the expression of genes involved in cell proliferation (Mao et al., 2005). It has also been shown that activation of mGlu5 receptor in mouse hippocampal slices results in phosphoinositide 3-kinase

(PI3K)-dependent AKT phosphorylation (Hou and Klann, 2004; Ribeiro et al., 2010).

The mechanism underlying mGlu5-dependent AKT activation has been elucidated, and

Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. MOL #104372 it has been reported that activation of mGluRs enhances formation of the complex mGlu5 receptor, Homer, and phosphoinositide 3-kinase enhancer (PIKE), which leads to activation of PI3K (Rong et al., 2003). PI3K increases the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3), facilitating the recruitment of pyruvate dehydrogenase kinase 1 (PDK1) and AKT to the cell membrane, where AKT is phosphorylated by PDK1 (Burgering and Coffer, 1995; Rong et al., 2003). AKT activation triggers important downstream signaling events, such as apoptosis inhibition Downloaded from and transcription activation of genes involved in cell proliferation and survival (Song et al., 2005). molpharm.aspetjournals.org

Various synthetic ligands were found to interact with mGlu5 receptor, facilitating the understanding of many different downstream signaling effects triggered by this receptor. 3,5-dihydroxyphenylglycine (DHPG) is an orthosteric ligand that can activate both mGlu5 and mGlu1 receptors (Fitzjohn et al., 1996; Ito et al., 1992). Further studies at ASPET Journals on September 26, 2021 led to the discovery of (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), which was at first propposed as a mGlu5 receptor selective agonist, but that was shown to also activate mGlu1 receptors (Doherty et al., 1997; Kammermeier et al., 2012). Another class of compounds that act as positive allosteric modulators (PAMs) can bind to an allosteric regulatory site that is topographically distinct from the conserved orthosteric agonist binding site, and thus can be more selective for the different mGluR subtypes since the allosteric site is weakly conserved (Sheffler et al., 2011). Interestingly, the mGlu5 receptor PAM, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), can activate neuroprotective cell signaling pathways without increasing intracellular

Ca2+ (Doria et al., 2013). Moreover, CDPPB can rescue neuronal cell death, abrogate huntingtin aggregation, and prevent memory deficit in a mouse model of Huntington’s disease (Doria et al., 2015). The widely used mGlu5 receptor inhibitor 2-methyl-6-

(phenylethynyl)-pyridine (MPEP) is a negative allosteric modulator (NAM), inhibiting the hydrolysis of phosphoinositides (Gasparini et al., 1999). MPEP can act as an inverse agonist of mGlu5 receptor, inhibiting its constitutive activity (Gasparini et al.,

1999; Pagano et al., 2000). In the same way, another potent selective mGlu5 receptor inhibitor is 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP), which shows higher affinity for the receptor and higher solubility in water in comparison to MPEP

(Cosford et al., 2003). Recent studies report that 2-chloro-4-((2,5-dimethyl-1-(4- Downloaded from (trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), a NAM with inverse agonist activity, is orally bioavailable and has in vivo affinity properties that are

30 to 100-fold higher than MPEP (Lindemann et al., 2011). Examples of mGlu5 molpharm.aspetjournals.org receptor synthetic ligands are presented in Table 1.

at ASPET Journals on September 26, 2021 Endocannabinoids and the CB1 receptor

The search for the mechanism of action of the main psychoactive component of the herb Cannabis sativa (hemp), Δ9-tetrahydrocannabinol (Δ9-THC), and its synthetic cannabinoids derivatives led to the discovery of the endogenous cannabinoid system

(Bisogno et al., 2005; Mechoulam and Gaoni, 1967; Paton, 1975; Wang and Ueda,

2009). The endocannabinoid system comprises at least two endogenous ligands

(endocannabinoids), the biochemical machinery for endocannabinoid synthesis and degradation, and the cannabinoid receptors, which include cannabinoid type 1 (CB1) receptor, CB2 receptor, and possibly others (Di Marzo, 2008; Di Marzo et al., 2004;

Mechoulam et al., 1995; Piomelli, 2003). CB1, a Gi/o-coupled receptor, is abundantly expressed in the mammalian CNS and is mainly located at the presynaptic sites of neurons of the basal ganglia, cerebellum, hippocampus, some regions of the cerebral

(Herkenham et al., 1990; Matsuda et al., 1990).

The major endogenous ligands of the cannabinoid receptors are arachidonoyl ethanolamide (AEA) (also termed anandamide) and 2-arachidonoyl glycerol (2-AG)

(Devane et al., 1992; Mechoulam et al., 1995). Besides AEA and 2-AG, other ligands have been proposed (Bisogno et al., 2000; Hanus et al., 2001; Huang et al., 2002; Porter

et al., 2002). Unlike other mediators, AEA and 2-AG are not stored in the CNS, but act Downloaded from as retrograde messengers that are synthesized and released in an activity-dependent manner, following changes in cellular homeostasis (Di Marzo and Matias, 2005; molpharm.aspetjournals.org Marsicano et al., 2003; Wilson and Nicoll, 2001). Increased Ca2+ levels can augment endocannabinoid synthesis, in part because their biosynthetic enzymes are sensitive to

Ca2+ concentration levels (Di Marzo, 2009).

Endocannabinoids are generally inactivated by mechanisms involving carrier- at ASPET Journals on September 26, 2021 mediated transport into the cell and intracellular hydrolysis (Piomelli, 2003). Because

AEA and 2-AG are lipophilic molecules, they can diffuse through the lipid membranes of both neurons and glia using carrier-mediated transport by facilitated diffusion

(Beltramo et al., 1997; Hillard et al., 1997; Piomelli, 2003). AEA is metabolized into arachidonate and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH) and

2-AG in arachidonate and glycerol via monoacylglycerol lipase (MAGL) (Di Marzo and

Petrosino, 2007; Piomelli, 2003). Evidence also suggest that FAAH can catalyze 2-AG hydrolysis (Bisogno et al., 2005; Di Marzo and Deutsch, 1998). Since FAAH is present at cell bodies and dendrites of principal neurons and MAGL is located at axon terminals, it has been proposed that AEA and 2-AG degradation occur at the post and presynaptic neuron, respectively (Dinh et al., 2002; Egertova et al., 2003; Tsou et al.,

1998).

CB1 receptor: Pharmacology and cell signaling pathways

CB1 receptor activation influences multiple effector systems, and due to its wide distribution in the brain, it is believed that CB1 receptor is responsible for mediating the majority of the cannabinoids effects in the CNS (Cabral and Marciano-Cabral, 2005; Di

Marzo, 2009). Evidence obtained by multiple groups support the hypothesis that the Downloaded from CB1 receptor plays a key role in various cellular events (Ferreira-Vieira et al., 2014;

Gomez et al., 2015; Zhang et al., 2015). Moreover, it has been reported that the CB1 receptor is involved in cognitive and affective processes, as well as in the control of molpharm.aspetjournals.org motor activity, stress, addiction and neuroprotection (Broadbent et al., 2004; Finn,

2010; McReynolds et al., 2016; Nagayama et al., 1999; Naydenov et al., 2014). Given its presynaptic location, it has been proposed that the CB1 receptor can regulate neurotransmitter release, and thus modulate synaptic plasticity by facilitating LTD at ASPET Journals on September 26, 2021

(Gerdeman et al., 2002; Marsicano et al., 2002; Robbe et al., 2002; Wilson and Nicoll,

2001). CB1 receptor activation effect on synaptic plasticity can occur through the

2+ inhibition of presynaptic voltage-gated Ca channels, including N- and P/Q-type channels, or by the activation of A-type and inward rectifying K+ channels (Felder et al.,

1993; Howlett et al., 2002; Piomelli, 2003; Twitchell et al., 1997; Wilson and Nicoll,

2002).

CB1 receptor stimulation activates multiple signal transduction pathways through the Gi/o family of G proteins (Fig. 2). Usually, CB1 receptor activation modulates both adenylate cyclase and mitogen-activated protein kinase (MAPK) activity. The classical effect of CB1 receptor agonist is the inhibition of adenylate cyclase activity and reduction of cAMP production, which has been demonstrated in several conditions

(Childers et al., 1994; Felder et al., 1993; Vogel et al., 1993). CB1 receptor agonists induce MAPK phosphorylation and MAPK activation, which suggests that the CB1 receptor can modulate gene transcription (Bouaboula et al., 1995; Patel et al., 1998).

Furthermore, MAPK regulates key cellular processes, such as cellular proliferation and survival that may be mediated by CB1 receptor (Aguado et al., 2006; Aguado et al.,

2007). Other effects attributed to CB1 receptor appear to involve activation of the

PI3K/AKT pathway (Molina-Holgado et al., 2002; Ozaita et al., 2007); recently, it was Downloaded from reported that activation of the anandamide/CB1/PI3K pathway protects rat brains from cocaine-induced neurotoxicity (Vilela et al., 2015). molpharm.aspetjournals.org Some evidence indicate that pharmacological manipulation of the cannabinoid system can allievate the sympotomology of several conditions, including addiction, seizures, neurodegenerative disease, and nociception (Adamczyk et al., 2009; Almeida-

Santos et al., 2013; Pietropaolo et al., 2015; Vilela et al., 2015; Wolkers et al., 2015). at ASPET Journals on September 26, 2021

For example, treatment with a CB1 receptor agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-4- morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212), prevented the appearance of motor deficits, decreased the number of striatal huntingtin inclusions, and rescued striatal medium-sized spiny neurons loss in a Huntington’s disease mouse model (Pietropaolo et al., 2015). Positive results have also been found in Alzheimer's and Parkinson's disease mouse models (Aso et al., 2012; Cao et al., 2014; Price et al., 2009). Others synthetic compounds, termed indirect agonists, are of therapeutic value for their ability to potentiate the action of endogenous ligands in a controlled manner by blocking metabolism or uptake of endocannabinoids. For instance, we have demonstrated that cyclohexyl carbamic acid

3'-carbamoyl-biphenyl-3-yl ester (URB597), a FAAH inhibitor, reduces cocaine- induced seizures and epileptiform electroencephalographic activity in rats (Vilela et al.,

2015). Similarly, 4-nitrophenyl-4-[bis(1,3-benzodioxol-5- yl)(hydroxy)methyl]piperidine-1-carboxylate (JZL-184, MAGL inhibitor) increases the levels of 2-AG, which reduces and delays the development of generalized seizures in a kindling model of temporal lobe epilepsy (von Ruden et al., 2015). Other effects, such as an increase in brain derived neurotrophic factor (BDNF) levels, cellular proliferation, and neurogenesis have also been reported with the use of cannabinoid receptors indirect agonists (Ferreira-Vieira et al., 2014; Gomez et al., 2015; Zhang et al., 2015). See Downloaded from Table 2 to for more examples of synthetic ligands of the endocannabinoid system.

molpharm.aspetjournals.org

The functional interaction between mGlu5 and CB1 receptors

mGlu5 and CB1 receptors exhibit a close relationship, as one receptor can regulate the activation of the other (Fig. 3). CB1 receptor activation leads to decreased at ASPET Journals on September 26, 2021 2+ Ca channel activity, which limits glutamate release and suggests that the CB1 receptor might regulate the amount of glutamate available to activate mGlu5 receptors

(Marsicano et al., 2003; Shen et al., 1996; Wilson and Nicoll, 2001). On the other hand, mGluR activation can facilitate endocannabinoid synthesis. For example, mGluR1 stimulation in cerebellar Purkinje cells can enhance endocannabinoid synthesis

(Maejima et al., 2001). Moreover, stimulation of group 1 mGluRs, especially the mGlu5 receptor, in hippocampal CA1 cells also leads to increased cannabinoid production (Ohno-Shosaku et al., 2002; Varma et al., 2001). A rise in postsynaptic Ca2+ due to neuronal stimulation increases cannabinoid synthesis as it can be prevented when calcium chelators, such as EGTA or BAPTA, are present postsynaptically (Llano et al.,

1991; Pitler and Alger, 1994). However, mGluR activation by DHPG can augment the depolarization-induced release of endocannabinoids without increasing intracellular

2+ Ca levels (Ohno-Shosaku et al., 2002). Thus, it is possible that the mGlu5 receptor might rely on other mechanisms to increase endocannabinoid synthesis. More recently, it has been demonstrated that the mGlu5 receptor is part of the 2-AG signalosome (Fig.

3), which was first identified at excitatory synapses of the ventral striatum and prefrontal cortex and was later found in other regions of the mammalian CNS (Jung et al., 2012; Katona et al., 2006; Matyas et al., 2008; Nyilas et al., 2009). This supra- molecular complex contains Homer proteins, which bind to mGlu5 receptor and to the Downloaded from two key enzymes responsible for 2-AG synthesis, PLC-β and DGL-α (Jung et al., 2007;

Jung et al., 2012; Katona et al., 2006). mGlu5 receptor stimulation leads to PLC-β

activation and formation of DAG, which can be rapidly converted into 2-AG due to the molpharm.aspetjournals.org close proximity of the enzyme DGL-α. As 2-AG signalosome is restricted to excitatory synapses, this increase in 2-AG synthesis mainly underlies a suppression of presynaptic glutamate release that can be important to prevent excitotoxicity, but can also decrease at ASPET Journals on September 26, 2021 excitability (Huang et al., 2008; Katona and Freund, 2008; Lafourcade et al., 2007).

CB1 and mGlu5 receptors mediate LTD:

LTD is a type of synaptic plasticity that likely contributes to learning and memory, and can be triggered by activation of NMDARs and mGluRs (Dudek and

Bear, 1992; Fitzjohn et al., 1999; Kemp and Manahan-Vaughan, 2004). LTD can be induced by DHPG and it has been shown that the mGlu5 receptor is the key mGlu to trigger LTD (Huber et al., 2001; Liu et al., 1993; Palmer et al., 1997). Notably, mGlu5 receptor stimulation can trigger LTD in both excitatory and inhibitory synapses by mobilizing endocannabinoids. It has been shown that repetitive activation of presynaptic glutamatergic fibers can induce mGlu-dependent long-term depression of hippocampal

Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. MOL #104372 inhibitory synapses in the CA1 area via retrograde cannabinoid signaling (Chevaleyre and Castillo, 2003). In addition, mGlu5-dependent LTD via mobilization of endocannabinoids, especially 2-AG, also takes place at excitatory synapses in the visual and prefrontal cortex (Huang et al., 2008; Lafourcade et al., 2007), as well as in the

Schaffer collateral pathway (Izumi and Zorumski, 2012). The mGlu5 receptor can also trigger LTD in a mechanism that is dependent on protein synthesis and might also involve CB1 receptor (Huber et al., 2002; Weiler and Greenough, 1993). This type of Downloaded from mGlu5/CB1-induced LTP will be further discussed at the section “CB1/mGlu5 role in

Fragile X syndrome”. The cooperative actions triggered by mGlu5 and CB1 receptors to

induce LTD indicates that these two receptors might be important in various molpharm.aspetjournals.org physiological processes, including memory and addiction. Indeed, CB1 and mGlu5 receptors appear to be involved in cocaine addiction, as a single exposure of mice to cocaine increases expression of Homer, reduces mGlu5 receptor plasma membrane at ASPET Journals on September 26, 2021 expression, and abolishes retrograde LTD mediated by endocannabinoids (Fourgeaud et al., 2004).

CB1 and mGlu5 receptors role in Fragile X syndrome:

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is caused by the silencing of the fragile X mental retardation 1 (FMR1) gene with consequent expression arrest of the fragile X mental retardation protein (FMRP), an RNA-binding protein important for translation regulation (Darnell et al., 2011;

Verkerk et al., 1991). It has been demonstrated that the mGlu5 receptor plays an important role in FXS (Bear et al., 2004; Michalon et al., 2012). Activation of group I mGluRs leads to protein-synthesis-dependent establishment of LTD, which is enhanced

Consistent with these studies, coupling between mGluR activation and endocannabinoid mobilization is enhanced at CA1 GABAergic synapses of FMR1 knockout mice, leading to augmented endocannabinoid-mediated LTD and increased neuronal excitability (Zhang and Alger, 2010). Similarly, in the FMR1 knockout mice dorsal striatum, mGlu5-mediated endocannabinoid activity at GABAergic synapses is increased (Maccarrone et al., 2010). Downloaded from

At excitatory synapses of the frontal cortex and ventral striatum, FMRP deletion promotes disorganization of the 2-AG signalosome, marked deficits in mGlu5-mediated molpharm.aspetjournals.org 2-AG release, and diminished endocannabinoid-dependent protein-synthesis- independent LTD (Jung et al., 2012). Homer binding to DGL-α is altered at excitatory synapses of FMR1 knockout mice, this leads to misplacement of DGL-α in the cell and disruption of the 2-AG signalosome, although no alterations in DGL-α localization were at ASPET Journals on September 26, 2021 observed at inhibitory synapses (Jung et al., 2012). Thus, LTD can be differentially regulated by FMRP, depending on whether it takes place at excitatory or inhibitory synapses. Supporting this hypothesis, when Homer interactions were disrupted by a peptide that binds to long Homers, hippocampal slices from both wild type and FMR1 knockout mice became more excitable due to enhanced mGlu5-dependent endocannabinoid mobilization at GABAergic synapses, although endocannabinoid mobilization by mGlu5 receptors was decreased at excitatory synapses (Tang and Alger,

2015). Interestingly, antagonism of CB1 receptor activity prevented the development of hyperexcitability in both FMR1 knockout and membrane-permeable Homer CT peptide models (Busquets-Garcia et al., 2013; Tang and Alger, 2015). However, pharmacological enhancement of 2-AG levels by JZL184 normalized eCB-mediated

LTD and corrected key behavioural abnormalities in FMR1 knockout mice (Jung et al.,

2012). Thus, at this point it is not clear whether CB1 receptor blockage or activation could be beneficial in FXS.

mGlu5 and CB1 receptors cooperatively modulate nociception:

The analgesic effect of cannabinoids in the CNS is mainly mediated by CB1 receptor expression in neural substrates involved in nociceptive neurotransmission, Downloaded from including the spinal dorsal horn, periaqueductal gray (PAG), dorsal raphe nuclei, and thalamic ventroposterolateral nucleus (Martin et al., 1995). Group 1 mGluRs can modulate nociceptive responses in the spinal cord, ventrobasal thalamus, dorsal raphe, molpharm.aspetjournals.org amygdala, and PAG (Hama, 2003; Palazzos et al., 2006; Young et al., 1997).

Importantly, it has been shown that mGlu5 and CB1 receptors exhibit a functional interaction to modulate nociception in some of these brain substrates. at ASPET Journals on September 26, 2021

The midbrain PAG is the most extensively studied supraspinal area involved in the modulation of nociception. Interestingly, it has been shown that the mGlu5 receptor is able to exert a tonic modulation of nociception promoted by microinjections of WIN

55,212-2 in the PAG, which increases nociceptive response latency in the plantar test (Palazzo et al., 2001). Moreover, under neuropathic pain conditions, PAG cannabinoid-induced antinociception requires downstream activation of the mGlu5 receptor (de Novellis et al., 2005; Palazzo et al., 2012). The mechanism underling mGlu5/CB1 cooperation at the PAG to promote analgesia has been identified, as it was demonstrated that mGlu5 receptor stimulation in PAG slices leads to 2-AG mobilization and retrograde inhibition of GABAergic transmission, which produces analgesia (Drew et al., 2008). Corroborating these data, microinjection of DHPG into the dorsolateral

PAG triggered the release of 2-AG and enhanced stress-dependent antinociception

(Gregg et al., 2012). This effect was reversed by pharmacologically blocking DAGLα or reducing expression through siRNA silencing (Gregg et al., 2012). Moreover, immunohistochemical analysis demonstrated that, at the PAG, the mGlu5 receptor colocalizes with DAGLα at the postsynaptic terminals in a location juxtaposed to the presynaptic localization of CB1 receptors, which suggests that postsynaptic mGlu5-

DAGLα cascade can trigger retrograde 2-AG signaling in the PAG to promote analgesia

(Gregg et al., 2012). Since the mGlu5 receptor is mostly present perisynaptically, Downloaded from spillover glutamate is the main endogenous mechanism to activate this receptor at the

PAG region (Drew et al., 2008; Nusser et al., 1994). For example, neuronal inhibition

of glutamate transporters in PAG slices enhanced mGlu5-mediated depression of molpharm.aspetjournals.org

GABAergic synaptic transmission through a CB1-dependent mechanism (Drew et al.,

2008). In addition, capsaicin and anandamide can activate TRPV1 and induce substantial glutamate release in the ventrolateral PAG to stimulate mGlu5-dependent 2- at ASPET Journals on September 26, 2021 AG mobilization and analgesia (Kawahara et al., 2011; Liao et al., 2011; Maione et al.,

2006; Starowicz et al., 2007). Moreover, several analgesic neuropeptides can trigger 2-

AG mobilization via mGlu5 receptor stimulation by exciting glutamatergic cell bodies.

It has been shown that Substance P, neurotensin, and cholecystokinin (CCK) can stimulate neurokinin 1, neurotensin 1/2, and cholecystokinin 1 receptors, respectively, on glutamatergic somas. This triggers glutamate release and leads to activation of perisynaptic mGlu5 receptors with consequent PAG disinhibition through 2-AG mobilization (Drew et al., 2009; Mitchell et al., 2011; Mitchell et al., 2009). Future studies will be important to determine whether this PAG disinhibition mechanism contributes to the antinociceptive effects of intra-PAG injection of substance P, neurotensin, and CCK.

As in the case of the PAG, a functional interaction between mGlu5 and CB1 receptors to modulate nociception takes place at the spinal cord and amygdala. It has been shown that the antihyperalgesic effect of WIN55,212-2 in rats submitted to unilateral loose ligation of a sciatic nerve is mediated through an interaction between

CB1 and mGlu5 receptors in the spinal cord dorsal horn (Hama and Urban, 2004).

Further studies demonstrated that DGL-α colocalizes with the mGlu5 receptor at the postsynaptic sites of nociceptive spinal synapses and also that the CB1 receptor is Downloaded from present at the presynaptic excitatory axon terminals (Nyilas et al., 2009). Moreover, intrathecal activation of mGlu5 receptor at the lumbar level evoked endocannabinoid-

mediated stress-induced analgesia through 2-AG mobilization, suggesting a key role for molpharm.aspetjournals.org

2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level

(Nyilas et al., 2009). mGlu5 and CB1 receptors also work cooperatively to promote analgesia in an arthritis pain model through a mechanism that involves CB1-mediated at ASPET Journals on September 26, 2021 increase in mGlu5 receptor function in the medial prefrontal cortex with consequent inhibition of abnormally enhanced amygdala output in pain (Ji and Neugebauer, 2014).

In addition, fear conditioning analgesia is facilitated by CB1 receptor activation in the basolateral amygdala via a mechanism that involves the modulation of mGlu5 and

GABA receptors signaling (Rea et al., 2013). Thus, mGlu5 and CB1 receptors work cooperatively in various brain substrates to modulate nociception.

Conclusion

The crosstalk between mGlu5 and CB1 receptors is important to regulate several physiological events, including LTD and neurotransmitters release. Furthermore, the mGlu5/CB1 signalosome complex appears to play a major role in some conditions, such as nociception and FXS, which indicates that these GPCRs are potential therapeutic

targets. After the discovery of the functional interaction between mGlu5 and CB1 receptors, several studies have emerged aiming to elucidate the mechanisms involved in this process. While it is clear that the formation of the signalosome containing mGlu5-

Homer-PLC-β-DGL-α is a key factor contributing to mGlu5-dependent mobilization of

2-AG and to the regulation of neurotransmission, more studies are needed to clarify the mechanisms and signaling pathways involved in this interaction. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Acknowledgments

The authors thank Roenick P. Olmo and Alvaro G. Ferreira for the insightful comments that significantly contributed to figures design.

Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Authorship Contribution

Wrote or contributed to the writing of the manuscript: Olmo, Ferreira-Vieira, Ribeiro. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

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Footnotes

IGO and THF contributed equally to this work. This work was supported by the

Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and the

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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molpharm.aspetjournals.org

at ASPET Journals on September 26, 2021

Figure legends

Figure 1: Cell signaling pathways activated by mGlu5 receptor

The scheme illustrates an overview of mGlu5 receptor cell signaling pathways.

Following glutamate binding, mGlu5 receptor is stimulated and promotes G-protein dissociation, which activates PLCβ and leads to DAG cleavage and formation of the Downloaded from second messengers IP3 and DAG. IP3 binds to and stimulates IP3Rs, increasing intracellular Ca2+ levels and contributing to ERK1/2 activation. In addition, the C-

terminal portion of mGlu5 receptor can interact with Homer proteins that interact with molpharm.aspetjournals.org other protein partners to trigger various biological cascades such as formation of the endocannabinoid 2-AG and phosphorylation of AKT and ERK1/2. Moreover, activation of these distinct pathways underlies important biological processes including cell proliferation and survival. Protein-protein interactions are represented by arrow- at ASPET Journals on September 26, 2021 chains and activated elements are represented by continuous arrows. Abbreviations: 2- arachidonoyl glycerol (2-AG); diacylglycerol (DAG); diacylglycerol lipase (DGL); extracellular signal-regulated kinases (ERK1/2); glutamate (Glu); inositol 1,4,5- triphosphate (IP3); inositol 1,4,5-triphosphate receptor (IP3R); metabotropic glutamate receptor (mGlu5); mitogen-activated protein kinase kinase (MEK); phosphoinositide 3- kinase (PI3K); phosphoinositide 3-kinase enhancer (PIKE); phospholipase Cβ (PLCβ).

Figure 2: Cell signaling pathways triggered by CB1 receptor activation

Activation of CB1 receptor by the endocannabinoids 2-AG and AEA can initiate a series of intracellular events mediated by the Gi/o protein. CB1 receptor activation can inhibit

adenylate cyclase activity and therefore modulate cAMP production. CB1 receptor activation also promotes closure of VGCC, which can reduce Ca2+ entry into the presynaptic neurons and interfere with synaptic vesicle exocytosis and neurotransmission. Furthermore, CB1 receptor stimulation can induce both MAPK and

AKT phosphorylation and activation, which regulate proliferation and cell survival.

Abbreviations: 2-arachidonoyl glycerol (2-AG); arachidonoyl ethanolamide (AEA); cannabinoid receptor type 1 (CB1); adenylate cyclase (AC); cyclic adenosine Downloaded from monophosphate (cAMP); adenosine monophosphate (AMP); voltage-gated Ca2+ channel (VGCC); mitogen-activated protein kinase (MAPK); calcium ion (Ca2+)

phosphoinositide 3-kinase (PI3K); Protein kinase B (AKT). molpharm.aspetjournals.org

Figure 3: Functional interaction between mGlu5 and CB1 receptors: The signalosome complex at ASPET Journals on September 26, 2021

At glutamatergic synapses, mGlu5 receptor intracellular domains interact with Homer protein, which is a scaffold protein important to assemble the complex known as signalosome. This signalosome complex, which is located at the perisynapse, brings mGlu5 receptor into close proximity to the two key enzymes responsible for 2-AG synthesis, PLC-β and DGL-α, as Homer proteins bind to mGlu5 receptor, PLC-β and

DGL-α. Thus, mGlu5 receptor stimulation leads to the activation of PLCβ and formation of IP3 and DAG, which is metabolized to 2-AG by DGLα. 2-AG moves across the synaptic cleft and activates CB1 receptor, located at the pre-synaptic terminal.

2+ Activation of CB1 inhibits VGCC, reducing the influx of Ca and, consequently, reducing glutamatergic vesicles exocytosis. The result of mGlu5 and CB1 receptors signaling crosstalk is the regulation of postsynaptic neurons excitability. Activated

Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. MOL #104372 elements are represented by continuous arrows, inhibition is represented by blocked arrows and reduced activity of biological process is represented by dashed arrow. The protein complex depicted in the dashed area, named as Signalosome Complex, is located at the plasma membrane of the perisynaptic region. Abbreviations: 2- arachidonoyl glycerol (2-AG); cannabinoid receptor type 1 (CB1); diacylglycerol lipase

α (DGLα); glutamate (Glu); ionotropic glutamate receptor (iGluRs); mitogen-activated protein kinase (MAPK); metabotropic glutamate receptor (mGlu5); phospholipase Cβ Downloaded from (PLCβ); voltage-gated calcium channel (VGCC); vesicular glutamate transporter 1

(vGLUT). molpharm.aspetjournals.org

at ASPET Journals on September 26, 2021

Tables

Table 1: Examples of mGlu5 receptor synthetic ligands

Chemical compound Target receptor References

mGlu1/5 3,5-dihydroxyphenylglycine (Ito et al.,

receptors Downloaded from (DHPG) 1992) agonist

molpharm.aspetjournals.org (RS)-2-chloro-5- mGlu5 receptor (Doherty et hydroxyphenylglycine agonist al., 1997) (CHPG)

at ASPET Journals on September 26, 2021 3-cyano-N-(1,3-diphenyl-1H- mGlu5 receptor (Kinney et pyrazol-5-yl)benzamide PAM al., 2005) (CDPPB)

2-Methyl-6-(phenylethynyl)- mGlu5 receptor (Gasparini pyridine NAM et al., 1999) (MPEP) (inverse agonist)

3-[(2-Methyl-1,3-thiazol-4- mGlu5 receptor (Cosford et yl)ethynyl]-pyridine NAM (inverse al., 2003) (MTEP) agonist)

2-chloro-4-((2,5-dimethyl-1-(4- mGlu5 receptor (trifluoromethoxy)phenyl)-1H- (Lindemann NAM (inverse imidazol-4-yl)ethynyl)pyridine et al., 2011) agonist) (CTEP)

Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Table 2: Examples of synthetic ligands of the endocannabinoid system

Chemical compound Target receptor References

arachidonyl-2- CB1 receptor (Hillard et chloroethylamide agonist al., 1999) (ACEA)

(R)-(+)-[2,3-dihydro-5-methyl- Downloaded from 3-(4-morpholinylmethyl) pyrrolo [1,2,3,-de]-1,4- CB1 receptor (Di Marzo, benzoxazin-6-yl]-1- agonist 2009) naphthalenylmethanone molpharm.aspetjournals.org mesylate (WIN 55,212-2)

1-(2,4-dichlorophenyl)-5-(4-

iodophenyl)-4-methyl-N-(1- CB1 receptor

(Di Marzo, at ASPET Journals on September 26, 2021 piperidyl)pyrazole-3- antagonist 2009) carboxamide (inverse agonist) (AM251)

Cyclohexyl carbamic acid 3'- (Mor et al., carbamoyl-biphenyl-3-yl ester FAAH inhibitor 2004) (URB597)

4-nitrophenyl-4-[bis(1,3- benzodioxol-5- (Long et al., yl)(hydroxy)methyl]piperidine- MAGL inhibitor 2009) 1-carboxylate

(JZL 184)

Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021 Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021 Molecular Pharmacology Fast Forward. Published on June 23, 2016 as DOI: 10.1124/mol.116.104372 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021