European Journal of Neuroscience

European Journal of Neuroscience, Vol. 39, pp. 334–343, 2014

doi:10.1111/ejn.12431

The molecular interplay between endocannabinoid and neurotrophin signals in the nervous system and beyond €kfelt,3 Tibor Harkany1,2 and Patrick Doherty4 Erik Keimpema,1,2 Tomas Ho

€g Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va 1:A1, SE-17177, Stockholm, Sweden 2 Department of Molecular Neuroscience, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090, Vienna, Austria 3 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 4 Wolfson Centre for Age-Related Diseases, King’s College London, London, SE1 9RT, UK 1

Keywords: brain development, growth factor, pain, sensory system, signalling interaction

Abstract Neurotrophins are traditionally known for their roles in neuronal development, function and survival. More recent data has highlighted the importance of neurotrophin signalling in adult signalling contexts, including the regulation of synaptic transmission. In addition, neurotrophin levels are increased in inflammatory and neuropathic pain leading to sensitization to painful stimuli. Endocannabinoid (eCB) signalling was initially studied in the context of synaptic transmission and pain alleviation whilst recently gaining attention due to its involvement in the development of the nervous system. Similar to neurotrophins, eCB levels also rise during pain perception but result in diminished pain sensations. The overlap of cellular functions between neurotrophins and eCB signalling leads to the hypothesis that these signalling systems are positioned to regulate each other and narrow the multitude of actions that both systems can promote to the specific need of the cell. Therefore, in this review, we examine to what extent the involvement of these two signalling systems is co-ordinated as opposed to being coincidental, and causal to neuronal circuit modifications in pain. Available data point to numerous direct molecular interactions between the neurotrophin and eCB signalling systems in developmental and adult contexts, including receptor-level interplay, transcriptional control and synergistic regulation of downstream signalling cascades. Although experimental observations specifically in pain circuits are limited, the universality of downstream signalling systems from both neurotrophin and endocannabinoid receptors suggest an interdependent relationship between these two diverse signalling systems.

Introduction The focus of this review is to survey existing literature on the molecular interplay between neurotrophins/growth factors and endocannabinoid (eCB) signalling systems, and in doing so to propose an interdependence between these systems as shown by cross-talk at the level of receptor or ligand expression and signal transduction mechanism, impacting the formation and adult function of the nervous system, particularly the molecular machinery required for synaptic neurotransmission. Recent data also underscore cross-modulation of the two signalling systems in pain processing and sensation. Our intent here is to highlight the fundamental reliance of sensory neuron networks on this molecular partnership.

Correspondences: Professor Patrick Doherty, 4Wolfson Centre for Age-Related Diseases, as above. E-mail: [email protected] Dr Erik Keimpema, 2Department of Molecular Neuroscience as above. E-mail: [email protected] Received 4 September 2013, revised 16 October 2013, accepted 18 October 2013

Neurotrophins: a molecular interface between inflammatory and neuronal responses Neurotrophins are a family of growth factors promoting neuronal development, function and survival (Lu et al., 2005), and classically include nerve growth factor (NGF; Levi-Montalcini & Angeletti, 1963), brain-derived neurotrophic factor (BDNF; Barde et al., 1982), neurotrophin-3 (NT-3; Maisonpierre et al., 1990b) and neurotrophin-4/5 (NT-4/5; Hallbook et al., 1991). Although other growth factors such as epidermal growth factor (EGF; Cohen, 1972) and fibroblast growth factor (FGF; Rudland et al., 1974) share functional similarities, we focus this review mainly on NGF and BDNF. NGF exerts many of its actions through the high-affinity tropomyosin kinase receptor A (TrkA). BDNF and NT-4/5 signal through the closely related TrkB receptor, while NT-3 binds to TrkC (Lewin & Barde, 1996). Neurotrophins can also interact with the low-affinity neurotrophin receptor (p75NTR), an important signalling node in its own right, but a rather enigmatic molecule that modulates the affinity and selectivity of TrkA signalling (Dechant & Barde, 1997). Conversely, it is also a component of a death-signalling receptor

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

Endocannabinoid and neurotrophin interactions 335 complex containing sortilin, a structurally distinct co-receptor (Jansen et al., 2007) which sensitizes p75NTR to pro-neurotrophins and promotes apoptosis (Jansen et al., 2007; Capsoni et al., 2010; Feng et al., 2010). Neurotrophins are produced and secreted by circumscribed subpopulations of neurons in the fetal and adult central nervous systems. Immune cells, such as mast cells (Leon et al., 1994) and macrophages (Krenz & Weaver, 2000), and Schwann cells (Singh et al., 1997; Krenz & Weaver, 2000) are the primary peripheral sources of neurotrophins. Neurotrophins have gained significant attention recently as a signalling node adjusting pain response thresholds via the regulation of effector systems. The importance of neurotrophin signalling in neuronal sensory circuitries underpinning pain transduction is stressed by the classification of sensory dorsal root ganglion (DRG) neurons based on the expression of Trk receptors (Ernfors et al., 1992; Molliver et al., 1995; Snider & Silos-Santiago, 1996), and the coincident increase in neurotrophin (Woolf et al., 1994; Lin et al., 2011) and Trk receptor levels (Lin et al., 2011) in inflammatory and neuropathic pain states, resulting in hyperalgesia (that is, increased sensitivity to painful stimuli) (Woolf et al., 1994). In addition to NGF’s developmental actions on DRG neurons (Crowley et al., 1994), it is recognised as a major modulator of pain. Seminal discoveries include high TrkA and p75NTR receptor expression on adult DRG neurons confined to the peptidergic (substance P and calcitonin gene–related peptide neuron population; McMahon et al., 1994; Bennett et al., 1996), and that NGF administration induces pain (Andreev et al., 1995). For example, cessation of NGF signalling by administration of a TrkA-IgG fusion construct reduced pain severity in a broad variety of experimental models of pain, particularly those associated with peripheral inflammation (McMahon et al., 1995; Koltzenburg et al., 1999). The increase in NGF levels in inflamed and damaged tissues (Brown et al., 1991; Leon et al., 1994; Santambrogio et al., 1994; Nilsson et al., 1997) triggers the excess activation of TrkA receptors on sensory neurons, which sensitizes transient receptor potential cation channel subfamily V member 1/vanilloid receptor 1 (TRPV1) through phospholipase C (PLC) gamma (PLCc) signalling pathways (Huang et al., 2006) (Fig. 1). TRPV1 channels are non-selective for mono- and bivalent cations, and are expressed by TrkA-containing nociceptors (Carlton & Coggeshall, 2001). Activation of TRPV1 channels is associated with increased sensitivity to noxious stimuli, including acid, heat and mechanical insults, and are thought to contribute to heat hypersensitivity in inflammatory pain (Walder et al., 2012). In addition to an indirect sensitization of TRPV1 channels to noxious stimuli on sensory neurons, NGF also can directly up-regulate TRPV1 channel expression trough TrkA receptors and increase its trafficking in nociceptors through mitogen-activated protein kinase (MAPK) p38 and phosphatidylinositide 3-kinase (PI3K)–Src kinase pathways (Ji et al., 2002; Zhang et al., 2005). Although p75NTR signalling does not have a direct effect on pain sensation, its activation leads to the sensitization of TrkA receptors to NGF (Zhang & Nicol, 2004) and thus to the sensitization of adult sensory neurons through mechanisms described above. The contribution of NGF to pain signalling seems to depend on the cell type affected and location of signalling. For example, and quite controversially, intrathecal injections of NGF improved homeostatic spinal conditions and preserved analgesic effects in a rodent model of neuropathic pain, suggesting a spatial organization of differential NGF-mediated control of pain signals (Cirillo et al., 2010). Hyperalgesia is also indirectly affected by NGF (Fig. 1), as this neurotrophin can stimulate mast cells to produce histamine (Stempelj

Fig. 1. Sensitization to pain through NGF signalling. Upon injury, mast cells and macrophages infiltrate the region and release pro-inflammatory mediators, including NGF. NGF induces hypersensitivity to stimuli (yellow bolt) through direct sensitization of TRPV1 channels (1) and induction of transcription resulting in an up-regulation of TRPV1 channels (2). The response to NGF can be potentiated by the ability of p75NTR to sensitize TrkA receptors (3) and a positive feedback loop on mast cells resulting in an increased production of NGF. Endocannabinoid production by mast cells and macrophages is not shown to increase visual clarity.

& Ferjan, 2005), metabolites of arachidonic acid (AA) such as prostaglandins (Marshall et al., 1999) and even NGF itself (Leon et al., 1994), to propagate a positive feedback loop (Marshall et al., 1990) facilitating immune cell infiltration and inflammation (Linker et al., 2009). eCBs (see below) are derivatives of AA and are involved in pain sensation and inflammation (Rice et al., 2002), suggesting a possible interaction between neurotrophins and endocannabinoid signalling.

Endocannabinoids: the neuroimmune basis of cannabis in pain management Δ9-Tetrahydrocannabinol (THC), the main psychoactive compound from Cannabis spp., has been used for centuries in the management of pain and inflammation (Di Marzo & Petrocellis, 2006). THC is traditionally known to engage type 1 cannabinoid receptors (CB1Rs) in the brain, and CB2Rs on immune cells at the periphery (Kano et al., 2009). However, recent data broaden this view by revealing functional CB2Rs in cortical (den Boon et al., 2012) and brainstem (Van Sickle et al., 2005) neurons. Conversely, CB1Rs have been implicated in the regulation of immune responses (Kaplan, 2013), adding additional layers of complexity to eCB modulation of pain signalling. Here, we will focus on canonical CB1R signalling and neurotrophin interactions in developing and adult neurons considering that experimental data only implicate CB1Rs in the development of neuronal networks so far (Keimpema et al., 2011). THC’s mechanism of action remained elusive until the advent of eCB research, particularly the discovery of cell-type-specific receptors and their discrete functions in decoding sensory information. The body’s own endogenous ligands for CB1R and CB2R, the eCBs, are small signalling lipids that broadly modulate synaptic neurotransmission in neuronal circuits including those that had evolved to process sensory information and painful stimuli (Kano et al., 2009). The first-described eCB, anandamide (AEA; Devane et al., 1992), is traditionally associated with analgesia (Richardson et al., 1998). Although AEA synthesis is rather incompletely understood it probably includes the hydrolysis of N-acylphosphatidylethanolamine (NAPE) precursors by NAPE-selective phospholipase D (NAPE-PLD) and/or ABHD4 (Ueda et al., 2005; Liu et al., 2008). Fatty acid amide hydrolase (FAAH) is the major enzymatic entity catalysing AEA degrada-

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 334–343

336 E. Keimpema et al. tion (Cravatt et al., 2001). FAAH manipulation either genetically or pharmacologically increases peripheral and central AEA levels, and promotes analgesia in a CB1R-mediated manner (Clapper et al., 2010). Although 2-arachidonoylglycerol (2-AG) was second to be identified as a putative eCB (Stella et al., 1997), it has eclipsed AEA in its perceived (patho-)physiological importance, partly due to the detailed description of its metabolic pathways that generate and terminate its action at cannabinoid receptors (Kano et al., 2009). 2-AG in vertebrates is synthesized through the conversion of an AA-containing diacylglycerol by sn-1-diacylglycerol lipase a or b (DAGLa/ b) depending on tissue type and maturity (Bisogno et al., 2003). 2-AG inactivation progresses primarily through hydrolysis by monoacylglycerol lipase (MGL; Long et al., 2009) with a lesser but nonetheless important contribution by the a/b-hydrolase domaincontaining six and 12 enzymes (ABHD6/12; Blankman et al., 2007; Marrs et al., 2010), generating AA and glycerol. Anandamide and 2-AG both contribute to pain through both CB1Rs and CB2Rs (Rice et al., 2002; Desroches et al., 2008; Kato et al., 2012). However, AEA itself, analogous to striatal neuronal networks, might also contribute indirectly to eCB-mediated pain sensation by regulating 2-AG bioavailability and physiological activity through activation of TRPV1 channels (Maccarrone et al., 2008). In addition to the direct involvement of 2-AG as a ligand in mediating analgesia (Desroches et al., 2008), the hydrolysis of 2-AG, as metabolic precursors, by MGL generates an AA pool that can drive neuroinflammation via the synthesis of prostaglandins, lipid mediators of inflammation (Nomura et al., 2011). Inhibition of MGL during neuroinflammation, that is, microglia and astroglia activation in the brain, inhibits the accumulation of proinflammatory interleukins and prevents microglial activation (Nomura et al., 2011), suggesting that this eCB can contribute to pain sensation and processing through both direct (ligand for CB1R/CB2R signalling) and indirect (metabolic precursor for inflammatory cytokines) mechanisms.

phages (Cantarella et al., 2011) and regulating their activity (Facci et al., 1995). The increased complexity of signalling pathways in the propagation of pain signals is stressed by the occurrence of both hyperalgesic (neurotrophins) and analgesic (eCBs) signalling systems in nociception. Another layer of complexity has been added by the recent description of interactions between neurotrophin and eCB signalling ranging from fetal development into adulthood in a wide variety of physiological processes, including pain (Farquhar-Smith et al., 2002; Keimpema et al., 2013).

Neurotrophin–eCB interactions in development The molecular pathways underlying neurotrophin–eCB actions are better understood in developmental contexts. For example, recent data on NGF–eCB interactions demonstrate that they can directly modulate each other’s functions. The CB1R is expressed by a wide range of developing neurons in their axons and growth cones, dynamic neurite tips that sense and respond to guidance cues (Hohmann & Herkenham, 1999; Mulder et al., 2008; Vitalis et al., 2008; Wu et al., 2010; Argaw et al., 2011; Keimpema et al., 2013; Simon et al., 2013). DAGLa is expressed mainly in growth cones while MGL accumulates in the stabilized axon segment (Mulder et al., 2008; Keimpema et al., 2013). The lack of MGL expression in the growth cone allows the production of focal 2-AG, in addition to

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Non-classical targets for AEA in sensory neurons In addition to acting at CB1R/CB2R receptors, AEA can directly interact with other receptors including the non-classical cannabinoid receptor GPR55 (Johns et al., 2007; Lauckner et al., 2008) and ion channels such as Ca2+, K+ and Na+ (Kano et al., 2009), and TRPV1 channels (Ross, 2003). Thus, although the analgesic effects of 2-AG and AEA (Rice et al., 2002) are thought to be predominantly mediated by CB1Rs on NGF-sensitive sensory neurons (Ahluwalia et al., 2002; Price et al., 2003), nociceptive excitatory presynapses in the dorsal horn (Nyilas et al., 2009), inhibitory spinal dorsal horn neurons (Pernia-Andrade et al., 2009) and in the brain (Tsou et al., 1998; Egertova & Elphick, 2000), care needs to be taken when interpreting data on AEA actions given their wider range of pharmacological lipid mediators of inflammation targets. Nonetheless, it is clear that activation of presynaptic CB1Rs results in the blockade of Ca2+ channels and subsequent inhibition of excitatory neurotransmission leading to a cessation of signal propagation (Stella et al., 1997; Strangman et al., 1998; Long et al., 2009; Schlosburg et al., 2010), and thus analgesia. In addition, CB1R signalling has also been implicated in hyperalgesia in chronic pain by reducing inhibitory c-aminobutyric acid (GABA) and glycine release from spinal interneurons (Pernia-Andrade et al., 2009), stressing the importance of cell-type specific eCB contributions to pain signalling. eCBs can also contribute indirectly to analgesia by their anti-inflammatory actions through CB2Rs on immune cells (Facci et al., 1995; Vincenzi et al., 2013), inhibiting the infiltration of mast cells and macro-

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Fig. 2. Endocannabinoid signalling in development and adulthood. (A) Focal 2-AG signalling in the growth cone, either paracrine from neighbouring cells or autocrine through synthesis by DAGLa within the growth cone, contributes to axonal growth and growth-cone steering decisions. While DAGLa accumulates in the growth cone, MGL is mainly expressed in the stabilized axon segment to prevent ectopic CB1R signalling (grey dashed line indicates disrupted 2-AG signalling in this domain). (B) When the growth cone reaches its postsynaptic target, MGL appears in the presynapse while DAGLa assumes its postsynaptic position. In this configuration, 2-AG synthesized at the postsynaptic site can bind to presynaptic CB1Rs resulting in the inhibition of neurotransmitter release (dark blue). In addition to presynaptic MGL, glia engulfing the synapse also express MGL (Tanimura et al., 2012) to prevent excess 2-AG escaping the synaptic cleft.

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 334–343

Endocannabinoid and neurotrophin interactions 337 eCB sources from neighbouring cells, to promote neurite outgrowth (Fig. 2). The presence of MGL in the stabilized axon prevents ectopic axonal CB1R signalling by the degradation of superfluous 2-AG. Thus, this configuration allows CB1R signalling to persist in a defined cellular compartment and limits off-target effects. It also introduces the notion of a ‘gate-keeper’ function for MGL; rather than simply terminating the action of 2-AG, MGL is actively preventing the inappropriate activation of CB1Rs to allow a fine-spatial compartmentalisation of eCB signalling in a polarised cell (Keimpema et al., 2011). Nerve growth factor stimulation in developing neurons resulted in the up-regulation of DAGLa, MGL and CB1R protein levels through a TrkA–PI3K-dependent pathway (Keimpema et al., 2013). Strikingly, MGL levels were not increased in the growth cones, stressing the importance of focal 2-AG signalling in motile neurite tips to promote neurite outgrowth (Keimpema et al., 2013). Inhibition of the proteasome, responsible for the degradation of proteins, resulted in a similar up-regulation of MGL, suggesting that NGF might regulate proteasomal degradation to control MGL levels. This finding was validated by the NGF-induced enhanced expression of breast cancer type 1 susceptibility protein (BRCA1), a caretaker protein involved in ubiquitination and proliferation (Atipairin et al., 2011). Upon inhibition of BRCA1, MGL levels increased in the growth cone of cholinergic neurons, limiting neurite outgrowth, suggesting that NGF can indeed promote eCB signalling by reinforcing the established signalling configuration (Keimpema et al., 2013), with MGL playing the important ‘gate-keeping’ role (Fig. 3A). Conversely, eCB signalling can also regulate NGF-mediated effects. In cholinergic neurons, NGF induced a multi-axonal phenotype that was pharmacologically corrected with a CB1R agonist, leading to enhanced neurite outgrowth (Keimpema et al., 2013). In addition, AEA and 2-AG inhibited the NGF-induced proliferation of human breast cancer cells through the suppression of TrkA receptors in a CB1R-mediated fashion (Melck et al., 2000). Although Melck and co-workers did not investigate whether the inhibitory effects of CB1R signalling on proliferation are due a shift towards differentiation, it appears that eCBs are capable of fine-tuning NGF-induced growth responses into more specific physiological processes. The finding that NGF itself up-regulates eCB signalling components to enhance focal signalling (Keimpema et al., 2013) reinforces the idea

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that NGF can locally sculpt its own effects by recruiting secondary signalling systems. Hippocampal cholecystokinin (CCK)-containing interneurons express high levels of CB1Rs in adulthood (Katona et al., 1999) and during development (Morozov et al., 2009). The importance of tightly regulated CB1R signalling for the migration of CCK+ interneurons is stressed by their misrouting upon in vivo THC administration during fetal development (Berghuis et al., 2005). As BDNF is required for the proper development of interneurons (Marty et al., 1996; Huang et al., 1999), it is likely that a molecular interplay exists, similar to NGF. Indeed, in purified CB1R+ interneuron cultures, AEA acted as a chemoattractant and regulated the migration of interneurons by transactivating TrkB receptors (Fig. 3B) (Berghuis et al., 2005). eCB signalling promoted the formation of CB1R-TrkB complexes and their transactivation relied on Src and Erk signalling cascades to promote interneuron migration (Berghuis et al., 2005). Furthermore, AEA was able to inhibit BDNF-induced neurite outgrowth and differentiation through Src and Erk second messengers, indicating that eCB signalling can hold off differentiation until interneurons reach their proper placement (Berghuis et al., 2005). Thus, similarly to NGF, eCBs can restrict the broad spectrum of BDNFmediated responses to ensure the proper spatiotemporal activation of specific physiological processes. The ability of eCBs to sculpt growth factor responses is further substantiated by the interplay with FGF signalling in axonal growth (Fig. 3C). FGF belongs to a family of heparin-binding growth factors, encompassing 23 members (FGF 1–23), which mediate their effects via five tyrosine kinase receptors (FGFR1–5; Beenken & Mohammadi, 2009). They regulate a variety of cellular processes (Blottner et al., 1989), including an important role during development and regeneration in the central and peripheral nervous system (Grothe & Nikkhah, 2001). Although little is known about the exact role of the FGF system in pain, promotion of astrocyte activity resulting in inflammation is considered to be a factor in nerve injury (Coyle, 1998; Madiai et al., 2003). There are interesting interactions between the FGF system and cannabinoids in the control of neurite outgrowth. Promotion of axonal growth involves several cell adhesion molecules (such as NCAM, N-cadherin and L1; Saffell et al., 1997) which activate FGF receptor signalling cascades (Williams et al., 1994a; Saffell et al., 1997). The requirement of DAGL activ-

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Fig. 3. Developmental neurotrophin–eCB interactions. (A) TrkA signalling by NGF (green triangle) upregulates the eCB signalling cassette through PI3K. This leads to a reduction in 2-AG signalling in the stabilized axon segment by increase MGL expression, and increased 2-AG availability in motile growth domains. This is maintained by the focal degradation of MGL by BRCA1 in the growth cone. (B) TrkB activation by BDNF (purple triangle) regulates CB1R and MGL mRNA transcription as well as increasing CB1R sensitivity to eCBs. BDNF-mediated eCB release may contribute to the latter phenomenon. (C) FGF (orange circle), binding to its cognate receptor (FGFR), triggers DAG production by phospholipase Cc (PLCc). PLCc-induced intracellular Ca2+ release has the potential to coincidently activate DAGLs to generate 2-AG, which can activate CB1Rs in an autocrine manner. Labels ‘e’ and ‘i’ refer to the extracellular and intracellular membrane surfaces, respectively. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 334–343

338 E. Keimpema et al. ity (Archer et al., 1999), and thus the synthesis of 2-AG, in FGFmediated neurite outgrowth suggests that CB1R signalling could act as a downstream signalling pathway (Archer et al., 1999; Williams et al., 2003). Indeed, CB1Rs were expressed on the growth cones of cultured cerebellar neurons and their activation induced neurite outgrowth regardless of FGF receptor inhibition (Williams et al., 2003). However, antagonism of CB1Rs prevented FGF-induced neurite outgrowth, reinforcing the position of CB1R signalling downstream of FGF receptors. In addition, the FGF-induced axonal growth response mediated by DAG hydrolysis relies on the activity of N- and L-type calcium channels in growth cones (Williams et al., 1994b; Archer et al., 1999). Activation of CB1Rs in the presence of N- and L-type calcium channel inhibitors blocked neurite outgrowth, strongly arguing that eCBs couple FGF receptor signalling to an axonal growth response (Williams et al., 2003). A similar example of growth factor interplay is the transactivation of epidermal growth factor receptors (EGFR) through CB1R stimulation in cultured corneal epithelial cells (Yang et al., 2010). Activation of CB1Rs resulted in EGFR phosphorylation and promoted cell proliferation and migration through Erk1/2, JNK1/2 and Akt-PI3K phosphorylation, associated with EGFR signalling (Yang et al., 2010). Similar to neurotrophins, EGF and FGF receptor signalling can interact with eCBs to promote specific processes resulting in axonal growth.

Neurotrophin–eCB interactions in the adult The presence of both hyperalgesic neurotrophin and analgesic CB1R signalling on sensory neurons (Ahluwalia et al., 2002; Price et al., 2003), as well as the localization of CB2Rs on immune cells (Munro et al., 1993; Facci et al., 1995), leads to our hypothesis that these systems can interact. Here, we will discuss recent evidence that not only supports this notion but also suggests an intimate interdependence between neurotrophin and eCB signalling. This includes mutual regulation of the expression of each others’ pathway components, direct cross-talk at the level of the expressed ligands and receptors, and synergistic regulation of downstream signalling cascades (Fig. 4A–D). The dynamic interplay between neurotrophins and eCB signalling is more and more recognized in pain sensation. For example, in a model of neuropathic pain, CB1Rs were up-regulated in the dorsal horn of the spinal cord and contributed to enhanced analgesic effects when treated with CB1R agonists (Lim et al., 2003). The up-regulation of CB1Rs was significantly decreased by pharmacological blockade of Trk receptors and extracellular signal-regulated kinase (Erk), implying a direct role for neurotrophin–Erk signalling in the regulation of CB1R signalling at the level of expression of the CB1R (Lim et al., 2003). Conversely, CB1R antagonism in diabetes-induced neuropathic pain restored an NGF deficit in diabetic mice and improved nerve fibre morphology (Comelli et al., 2010), fuelling the concept that these systems exert reciprocal influences on one another. The interaction between eCBs and neurotrophins in pain signalling is further emphasized by the reduction in NGF-mediated hyperalgesia by the administration of AEA in an inflammatory pain model in rats (Farquhar-Smith et al., 2002; Farquhar-Smith & Rice, 2003). The molecular mechanisms of eCB-mediated prevention of NGF-induced hyperalgesia point in the direction of TRPV1 channels. In neurophysiological studies, exogenous NGF sensitizes TRPV1 signalling in sensory neurons to capsaicin (Evans et al., 2007; McDowell et al., 2013). Treatment with ACEA, a CB1R-specific agonist, prevented the sensitization of TRPV1 channels (McDowell et al., 2013) in a dose-dependent manner (Evans et al., 2007), suggesting that the analgesic effects of CB1R signalling are at least

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Fig. 4. Schematic overview of similarities between neurotrophin receptors and CB1R second = messenger pathways. (A and B) A simplified schema of second messengers illustrates that TrkA and TrkB signalling share many commonalities between their second-messenger pathways that regulate growth responses, survival and cytoskeletal modifications. Importantly, the actual pathway used can vary between different neuronal types. (C) In addition, FGFRs, molecularly distinct from neurotrophin receptors, can recruit similar signalling mediators to promote growth and survival. (D) The CB1R is a G-protein-coupled receptor that probably forms homodimers or multimers as signalling units (a single receptor is shown for clarity). CB1Rs signal through Gbc and Ga subunits to second messengers similar to those recruited by growth factor signalling. Thus, signalling interactions could occur between growth factor receptors and CB1Rs downstream of the receptor to fortify common second-messenger cascades to modulate a physiological response. Labels ‘e’ and ‘i’ refer to the extracellular and intracellular membrane surfaces, respectively.

in part contributing to the regulation of NGF-induced TRPV1 channel sensitivity. While the available data on the interactions between eCBs and NGF in adult signalling systems are sparse, the molecular dynamics between BDNF and eCBs are better understood. BDNF is a widely expressed (Maisonpierre et al., 1990a) activity-regulated neurotrophin (Zafra et al., 1990) and promotes neural survival (Barde et al., 1982; Davies et al., 1986), migration (Polleux et al., 2002), differentiation (Kalcheim et al., 1987; Sieber-Blum, 1991) and synaptogenesis (Seil & Drake-Baumann, 2000). BDNF signals through TrkB receptors (Squinto et al., 1991) and acts as a potent regulator of synaptic plasticity in the adult (Seil & Drake-Baumann, 2000). CB1Rs are involved in the developmental processes (Harkany et al., 2008) and synaptic plasticity (Kano et al., 2009) of neurons that respond to BDNF, e.g. glutamatergic pyramidal cells (Horch et al., 1999), GABAergic interneurons (Marty et al., 1996; Berghuis et al., 2006) and sensory neurons (Jones et al., 1994). BDNF is, in contrast to NGF, synthesized under physiological conditions in a subpopulation of mainly TrkA-positive DRG neurons (Michael et al., 1999). Upon peripheral nerve injury, BDNF is upregulated in these TrkA cells. Moreover, long-lasting, axotomy-induced up-regulation of BDNF mRNA and protein has been demonstrated in large-diameter TrkB- and TrkC-positive DRG neurons (Michael et al., 1999; Merighi et al., 2008). In addition, BDNF and TrkB are both also up-regulated in inflammation (Lin et al., 2011). Cell-type-specific

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 334–343

Endocannabinoid and neurotrophin interactions 339 deletion of BDNF from nociceptors ameliorates NGF-induced hyperalgesia (Zhao et al., 2006), suggesting a complex interplay between different neurotrophins in pain sensation. This, together with the similarities in the activity-dependent regulation (Zafra et al., 1990) between NGF and BDNF, provoked research into the possible interactions between BDNF and eCB signalling. Indirect evidence of a possible interaction came from studies focussing on the effects of CB1R signalling on BDNF levels in the context of addiction biology and behavioural neuroscience. Firstly, in a double-blind, placebo-controlled study (D’Souza et al., 2009), THC increased BDNF levels in the serum of healthy human subjects. Secondly, chronic THC administration led to the accumulation of BDNF mRNA and protein in brain centres related to addiction and reward, including the nucleus accumbens and to a lesser extend the ventral tegmental area, paraventricular nucleus and medial prefrontal cortex, in adult male rats (Butovsky et al., 2005). As BDNF is a regulator of drug dependence (Ghitza et al., 2010), the addictive effects of cannabis, though controversial, are enticing as contributors to increased BDNF signalling. In line with these findings are diminished levels of BDNF in the hippocampus of mice upon pharmacological inhibition of CB1Rs (Beyer et al., 2010). In addition, BDNF levels are decreased in CB1R-null mice (Aso et al., 2008) and in mice with a specific deletion of CB1Rs in principal neurons in the forebrain (Marsicano et al., 2003). Administration of BDNF in the hippocampus reversed enhanced stress responses in CB1R knockouts, implying a CB1R-mediated control of BDNFinduced synaptic plasticity in emotional responses (Aso et al., 2008). Although these data suggest an interaction, more compelling evidence is provided by studies on CB1R expression and function by BDNF itself. For instance, administration of BDNF to cultured cerebellar granule neurons up-regulates CB1R mRNA while decreasing MGL mRNA. This new configuration sensitizes cerebellar granule neurons to 2-AG concentrations otherwise not biologically active (Maison et al., 2009). Vice versa, in vivo THC administration increases hippocampal BDNF mRNA levels in an Erk-dependent manner (Derkinderen et al., 2003). In addition, in an in vitro model of Huntington disease, CB1R signalling enhanced the expression of both CB1R and BDNF mRNA (Laprairie et al., 2013). Neurophysiological studies in the cerebral cortex revealed that application of BDNF reduces the amplitude of inhibitory postsynaptic currents and decreases the release probability of GABA (LemtiriChlieh & Levine, 2010; Nieto-Gonzalez & Jensen, 2013). This effect was fully blocked by CB1R antagonists, suggesting that BDNF triggers eCB release from postsynaptic terminals to facilitate retrograde signalling (Lemtiri-Chlieh & Levine, 2010). However, application of BDNF in the visual cortex and striatum prevented CB1R activity at GABAergic synapses (Huang et al., 2008; De Chiara et al., 2010) indicating that the control of eCB signalling by BDNF is a context-dependent signalling system. While these studies provide a basis for the interplay between two signalling systems, the role of TrkB (similar to NGF receptors) and the underlying molecular mechanisms at the synaptic level remain unclear. Unfortunately, less is known of the molecular interactions between eCB and neurotrophin signalling in the adult. There is a particular lack of knowledge on the control of enzymes regulating eCB synthesis and the role of receptor expression patterns and receptor interactions. However, recent data on the developmental interactions between neurotrophins and eCBs suggest that interactions will exist in the adult, and shed some light on the potential nature of these interactions. Therefore, similar interactions may be important for pain networks where CB1Rs and Trk receptors are

co-expressed on sensory neurons, spinal cord and brain centres regulation pain perception (Ahluwalia et al., 2002; Price et al., 2003).

Neurotrophin–eCB interactions in pain signalling: an outlook Neurotrophic factors have a broad range of physiological effects, ranging from development until adulthood. Therefore, it is crucial for the cell to recognize which behaviour is preferential when confronted by a multi-faceted signalling molecule. Similar to neurotrophins, eCBs are involved in analogous physiological processes depending on the enzymatic distribution of the eCB signalling unit (receptors and synthesis/degradation enzymes) and the spatiotemporal position of the cell. Therefore, eCBs are well positioned to limit the global actions of a neurotrophic factor to a contextual relevant cellular response. Indeed, recent data on the interplay between neurotrophic factors and eCBs highlight a bidirectional regulation through shared second-messenger systems with often opposing outcomes, implying a tightly regulated signalling mechanism relevant to the spatiotemporal position of the cell. In addition, this interaction can be found in a wide variety of tissues from fetal development until adulthood, as well as with other growth factors and growthpromoting interleukins (Zorina et al., 2010), indicating that this molecular interplay is more the rule than the exception. The above is especially the case for pain research, where neurotrophins and eCBs are involved in similar physiological processes but are mostly investigated as singular signalling entities. The critical role for neurotrophins and CB1R function in pain sensation (Andreev et al., 1995; McMahon et al., 1995; Agarwal et al., 2007), together with the interplay between Trk receptors and CB1Rs (Lim et al., 2003), stresses the importance of a neurotrophin–eCB axis in the control of pain. The widespread presence of neurotrophin–eCB interactions in developmental processes, as described above, argues for similar undiscovered interplays in the establishment of pain circuitry and requires a change of focus to investigate these molecular interactions to fully understand the complex signalling systems involved in the establishment and function of pain circuitries.

Acknowledgements This work was supported by the Swedish Medical Research Council (T.Ha., T.H€ o.), Swedish Brain Foundation (‘Hj€arnfonden’; T.Ha.), Novo Nordisk Foundation (Nordic Endocrinology Research Initiative; T.H., T.H€ o.), the Petrus & Augusta Hedlunds Foundation (T.H., T.H€ o.), National Institutes of Health (RO1-DA023214; T.Ha.), European Commission, PAINCAGE project (T.H.) and the Wellcome Trust (P.D.).

Abbreviations 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; ABHD4/6/12, a/b hydrolase domain-containing proteins 4, 6 or 12; AEA, anandamide; BDNF, brain-derived neurotrophic factor; CB1R, type 1 cannabinoid receptor; CCK, cholecystokinin; DAGLa/b, sn-1-diacylglycerol lipase isoforms a/b; DRG, dorsal root ganglion; eCB, endocannabinoid; EGF, epidermal growth factor; Erk, extracellular signal-regulated kinase (1/2); FAAH, fatty acid amide hydrolase; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GABA, c-aminobutyric acid; MGL, monoacylglycerol lipase; NAPE, N-acyl phosphatidylethanolamine; NGF, nerve growth factor; p38 MAPK, p38 mitogen-activated protein kinase; p75NTR, low-affinity nerve growth factor receptor; PI3K, phosphatidylinositol 3-kinase; PLC, phospholi9 pase C; THC, D -tetrahydrocannabinol; TrkA or B, tropomyosin kinase receptor A or B; TRPV1, transient receptor potential cation channel subfamily V member 1/vanilloid receptor 1.

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