REVIEW / SYNTHESE

Role of the Go/i signaling network in the regulationof neurite outgrowth1

John Cijiang He, Susana R. Neves, J. Dedrick Jordan, and Ravi Iyengar

Abstract: Neurite outgrowth is a complex differentiation process stimulated by many neuronal growth factors and trans-mitters and by electrical activity. Among these stimuli are ligands for G-protein-coupled receptors (GPCR) that function asneurotransmitters. The pathways involved in GPCR-triggered neurite outgrowth are not fully understood. Many of these re-ceptors couple to Gao, one of the most abundant proteins in the neuronal growth cones. We have studied the Go signalingnetwork involved in neurite outgrowth in Neuro2A cells. Gao can induce neurite outgrowth. The CB1 cannabinoid recep-tor, a Go/i-coupled receptor expressed endogenously in Neuro2A cells, triggers neurite outgrowth by activating Rap1,which promotes the Gao-stimulated proteasomal degradation of Rap1GAPII. CB1-receptor-mediated Rap1 activation leadsto the activation of a signaling network that includes the small guanosine triphosphate (GTP)ases Ral and Rac, the proteinkinases Src, and c-Jun N-terminal kinase (JNK), which converge onto the activation of signal transducer and activator oftranscription 3 (Stat3), a key transcription factor that mediates the gene expression process of neurite outgrowth inNeuro2A cells. This review describes current findings from our laboratory and also discusses alternative pathways that Go/i

might mediate to trigger neurite outgrowth. We also analyze the role neurotransmitters, which stimulate Go/i to activate acomplex signaling network controlling neurite outgrowth, play in regeneration after neuronal injury.

Key words: G protein, cell signaling, neurite outgrowth, cannabinoid receptor.

Resume : L’excroissance des neurites est un processus de differenciation complexe stimule par de nombreux transmetteurset facteurs de croissance neuronale ainsi que par l’activite electrique. Parmi ces stimuli, les ligands des recepteurs couplesaux proteines G (RCPG) agissent comme des neurotransmetteurs. Les voies intervenant dans l’excroissance des neuritesdeclenchee par les RCPG ne sont pas totalement elucidees. Nombre de ces recepteurs se couplent a la proteine Gao, l’unedes proteines les plus abondantes dans les cones de croissance. Nous avons examine le reseau de signalisation de Go inter-venant dans l’excroissance des neurites dans les cellules Neuro-2A. Gao peut induire l’excroissance des neurites. Le recep-teur cannabinoıde CB1 (RCB1), un recepteur couple a Go/i, exprime de maniere endogene dans les cellules Neuro-2A,declenche l’excroissance des neurites par l’activation de Rap1 en favorisant la degradation proteasomale de Rap1GAPIIstimulee par Gao. L’activation de Rap1 vehiculee par RCB1 conduit a l’activation d’un reseau de signalisation constituedes petites GTPases Ral et Rac, des proteines kinases Src et c-Jun N-terminal (JNK) qui convergent vers l’activation dutransducteur de signal et de l’activateur de transcription 3 (Stat3), un facteur de transcription cle qui vehicule le processusd’expression genique de l’excroissance des neurites dans les cellules Neuro-2A. Cette synthese decrit les resultats actuelsde notre laboratoire et discute des voies que Go/i pourrait vehiculer pour declencher l’excroissance des neurites. Nous ana-lysons aussi comment les neurotransmetteurs qui stimulent Go/i pour activer un reseau de signalisation complexe regulantl’excroissance des neurites peuvent jouer un role important dans le regeneration apres une lesion neuronale.

Mots cles : proteine G, signalisation cellulaire, excroissance des neurites, recepteur cannabinoıde.

[Traduit par la Redaction]

Introduction

The differentiation process in neurons is a complex phe-nomenon involving changes in electrophysiological proper-ties and morphological features, characterized by dentriticand axonal outgrowths, broadly termed neurite outgrowth(Larkum et al. 1999). The regulation of neurite outgrowth istightly controlled because of its critical physiological func-tion. Multiple signals control the growth and directionalityof dendrites and axons (Song and Poo 1999; Yuan et al.2003). Many intracellular signaling components that controlneurite outgrowth have been studied (Xiang et al. 2002);however, their connections and relative positions within net-

Received 29 August 2005. Published on the NRC ResearchPress Web site at http://cjpp.nrc.ca on 23 August 2006.

J.C. He,2,3 S.R. Neves, J.D. Jordan, and R. Iyengar.Department of Pharmacology and Biological Chemistry, MountSinai School of Medicine, One Gustave L levy Place, NewYork, NY 10029, USA.

1This paper is one of a selection of papers published in thisSpecial issue, entitled Second Messengers andPhosphoproteins—12th International Conference.

2Corresponding author (e-mail: Cijiang.he@mssm.edu).3Present address: Department of Medicine, Mount Sinai Schoolof Medicine, One Gustave L levy Place, New York, NY 10029,USA.

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works have not been fully defined. Ligand-dependent activa-tion of several Go/i-protein-coupled receptors has been impli-cated in the regulation of neurite outgrowth. For example,D2 dopamine receptors regulate neurite outgrowth in corti-cal neurons (Reinoso et al. 1996). Serotonin-1 B receptorsare known to enhance neurite outgrowth in thalamic neurons(Lotto et al. 1999). Gao, one of the most abundant proteinsin the neuronal growth cones, can induce neurite outgrowthupon activation (Strittmatter et al. 1990, 1994). However,the signaling pathways through which the Gai/o signals trig-ger neurite outgrowth have not been determined.

Recently, we demonstrated that the CB1 cannabinoid re-ceptor, a Gai/o-coupled receptor, which is endogenously ex-pressed in Neuro2A cells, mediates neurite outgrowth byactivating the small G protein Rap1. The underlying mecha-nism involves the enhancement of the proteasomal degrada-tion of Rap1GAPII, a guanosine triphosphate (GTP)aseactivating protein for Rap1 (Jordan et al. 1999, 2005). Fur-thermore, we found that CB1 receptor stimulation leads tothe activation of a network that includes other smallGTPases, such as Ral and Rac, the protein kinases Src, andc-Jun N-terminal kinase (JNK). This network converges onsignal transducer and activator of transcription (Stat)3,which appears to be the critical component in the process ofneurite outgrowth (He et al. 2005). In this review, we willsummarize the downstream signaling pathways of Go/i anddiscuss the role of these signaling molecules in controllingneurite outgrowth and their possible implication in neuronalregeneration.

Go/i signaling network in neuronal cells

Many hormones, sensory stimuli, and neurotransmittersuse heterotrimeric G proteins to convert extracellular signalsinto active intracellular signaling networks, resulting in acellular response (Gilman 1987; Rodbell 1980). Heterotri-meric G proteins are composed of a, b, and g-subunits. Theb- and g-subunits are considered to be a single functionalcomplex because they do not dissociate in nondenaturingconditions. The a-subunit has the ability to bind and hydro-lyze GTP. In the basal state, the a-subunit is bound to gua-nosine diphosphate (GDP) and associates with the bgcomplex. Upon ligand binding to the G-protein-coupled re-ceptor, the a-subunit undergoes a conformational change,such that it promotes the exchange of GDP for GTP. In theGTP-bound state, the a-subunit dissociates from the bgcomplex, and both the a-subunit and the bg complex canthen interact with and regulate downstream effectors toevoke physiological responses (Cabrera-Vera et al. 2003;Offermanns 2003; Nguyen and Iyengar 2005). The a-subunithas intrinsic GTPase activity, leading to the eventual hydrol-ysis of the bound GTP to GDP, terminating the signal andallowing trimerization with the bg complex.

The heterotrimeric G-protein superfamily contains manymembers. There are at least 16 genes for the a-subunit (Hur-owitz et al. 2000). The a-subunits have been arranged into 4functional families: Gas, Gai/o, Gaq, Ga12/13. The Gai/o familyis comprised of Gai1, Gai2, Gai3, Gao, Gaz, Gagust, Gat-r, andGat-c. This family, with the exception of Gaz, is pertussis-toxin sensitive. Pertussis toxin inhibits Gai/o by promotingthe adenosine-diphosphate ribosylation of the COOH-

terminus of the G-protein a-subunit, thus preventing its in-teraction with the receptor. Gai has ubiquitous tissue distri-bution. Gao expression is limited to the brain, suggesting animportant role in neuronal signaling. Because it is difficultto distinguish functionally between Gai and Gao down-stream signaling pathways, most people refer Gai/o together.

Because the most abundant G protein in the mammalianbrain is Gao, our discussion on neurite outgrowth focuseson the Gao pathway. In contrast to the classic heterotrimericGas pathway, the downstream signaling pathway of Gao re-mains to be completely elucidated. In the Go/i pathway, boththe a-subunit and the bg complex can modulate downstreameffectors. The effectors directly regulated by Gbg includePLC-b, K+ channels, adenylyl cyclase, and phosphatidylino-sitol 3-kinase (PI3K) (Neves et al. 2002). For last severalyears, our laboratory and others have been studying down-stream components of Gao to explore its signaling path-ways. It has been reported that, in NIH3T3 cells,constitutively active Gao causes the activation of Src andStat3 (Ram et al. 2000). It has also been reported that Gai2mediates the activation of Stat3 (Corre et al. 1999), but themechanism of Src activation by Gao was not addressed inthat study. Using a combination of yeast 2-hybrid screensand heterologous-expression experiments, we found proteinsthat are direct candidate effectors of Gao. They include theGTPase activating protein (GAP) for the small G protein Rap(RapGAP), the GAP from Gaz (Gaz-GAP), the regulator of Gprotein signaling 17 (RGS-17), and the G-protein-regulatedinducer of neurite outgrowth (GRIN) (Jordan et al. 1999;Chen et al. 1999). The various pathways regulated by Gai/oare shown in Fig. 1. Here, we focus on how Gao causes theactivation of Rap1 (Jordan et al. 1999, 2005).

Rap1GAPII is the GTPase activating protein of thesmall protein Rap1. Studies have shown that Gao signal-ing can promote the ubiquitination and proteasomal degra-dation of Rap1GAPII. We found that the overexpressionof Gao reduced the protein stability of Rap1GAPII,whereas the presence of proteasomal inhibitors, such aslactacystin, increased protein levels (Jordan et al. 2005).In vitro and in vivo experiments have confirmed the inter-action and have shown that Rap1GAPII preferentiallybinds to the inactive Gao (Jordan et al. 2005). Increaseddegradation of Rap1GAPII leads to enhanced Rap activity(Jordan et al. 2005). Others have also shown that Rap1-GAP can be ubiquinated and degraded by the proteasomein a GSK3b-phosphorylation-dependent manner (Tsygan-kova et al. 2004).

Go/i signaling pathways in neurite outgrowth

Neurite outgrowth is a process by which neurons achieveconnectivity during brain development. The mechanismunderlying this process requires the coordination of signalscoming from outside and inside the cell. A critical structurein the neurite that might be important for such a function isthe growth cone. The growth cone is the motile structure atthe tip of elongating axons and dendrites; it is thought to beresponsible for recognizing pathways and targets and trans-ducing such information into directed movement (Stritt-matter and Fishman 1991). Growing axons are guided to

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appropriate targets by the responses of their motile growthcones to environmental cues.

Many signals participate in the regulation of neuronal out-growth, and evidence suggests that heterotrimeric G-proteinpathways play an important role. Gao accounts for 1% of allmembrane proteins in the brain, and is highly enriched ingrowth-cone membranes (Strathmann et al. 1990; Exner etal. 1999; Strittmatter et al. 1990). Therefore, many investi-gators have studied this G protein to determine its signifi-cance in neurite outgrowth. It has been shown that thecollapse of growth cones is mediated by G proteins; pertus-sis toxin inhibits growth-cone collapse (Igarashi et al. 1993).In addition, the expression of constitutively active Gao re-sults in an increase in the number of neurites per cell (Stritt-matter et al. 1994). However, little else is known about theway Gao affects neurite outgrowth. During the past fewyears, our laboratory has focused on investigating the down-stream effectors of Gao signaling for neurite outgrowth. Thefindings are described below and summarized in Fig. 2.

Role of small GTPasesSmall GTPases that play a major role in actin organiza-

tion have been found to be involved in the regulation neuriteoutgrowth. Gao activates Rap1 in Neuro2A cells by reducingthe amount of Rap1GAPII via ubiquitination and proteaso-

mal degradation pathway. We found that Gao-induced neu-rite outgrowth was inhibited by blocking Rap1 activity,using a dominant negative mutant. We have also testedthis hypothesis in an endogenous system. The CB1 recep-tor, a Gai/o-coupled receptor, is endogenously expressed inNeuro2A cells. Hu-210, a high-affinity ligand for the CB1receptor, stimulates neurite outgrowth in Neuro2A cells.Dominant negative Rap1 and siRNA of Rap1 inhibitedHu-210-induced neurite outgrowth, providing evidence of arole for Rap1 in receptor-induced neurite extension. In ad-dition, both Hu-210- and Gao-induced neurite outgrowthwas suppressed by lactacystin, an inhibitor of proteasomaldegradation (Jordan et al. 2005). These results suggest thatthe CB1 receptor, by regulating the proteasomal degrada-tion of Rap1GAPII, activates Rap to induce neurite out-growth. Current studies indicate that Gai/o subunitsstimulate the ubiquitination of Rap1GAPII. The precisemechanism by which this happens is not known. It is pos-sible that Gao, by binding to Rap1GAPII, facilitates itsubiquitination with an E3 ligase. However, other mecha-nisms are also possible. These mechanisms cannot be de-fined until the E3 ligase responsible for Gao-stimulatedubiquitination of Rap1GAPII is identified.

Recent studies have identified a potent Rho GAP, RA-RhoGAP, as a direct downstream target of Rap1 in the neu-

Fig. 1. Signaling through the Go/i pathway. Many neurotransmitters, hormones, and chemokines signal through Go/i-coupled receptors. Thepathways that are activated by both the Gbg and Ga subunits, and the cellular machines they regulate, are schematically depicted. GIRK, G-protein-coupled inwardly rectifying potassium channel; MAPKs, mitogen-activated protein kinases; Rap1-GAP, guanosine triphosphate(GTP)ase activating protein; GRIN, G-protein-regulated inducer of neurite outgrowth; RGS, regulator of G-protein signaling.

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rite outgrowth (Yamada et al. 2005). Overexpression of RA-RhoGAP induced the inactivation of Rho and promoted theneurite outgrowth in a Rap1-dependent manner. Althoughwe have not explored the Rap to Rho connection, such apathway could be critical in coordinating the cytoskeletal re-arrangement with the changes in gene expression that shouldoccur during neurite outgrowth.

Hu-210 activates Ral via Rap1 in Neuro2A cells (He et al.2005). Dominant negative Ral abolished receptor-inducedneurite outgrowth, indicating a role for Ral in Gao-mediatedneurite outgrowth. The role of Ral in neurite outgrowth ap-pears to be contextual. In the Gao–Rap pathway, Ral stimu-

lates neurite outgrowth. However, in PC12 cells, Ral-GEFs(Ral-guanine nucleotide exchange factors) and Ral activa-tion inhibits Ras-mediated neurite outgrowth (Goi et al.1999). This inhibition might be due to the interaction of Ralwith Ral-GDS (Ral-guanine nucleotide dissociation stimula-tor), which also functions in the Ras pathway. These datasuggest that Ral increases the Rap1 signal but counteractsthe Ras signal, and thus serves as a tuning mechanism forselection between upstream GTPases.

We also found that Rac1 is involved in receptor- andGao-induced neurite outgrowth. Dominant negative Rac1blocked Hu-210-induced neurite outgrowth in Neuro2Acells. Rac and its downstream effectors, PAK family kin-ases, have been shown to regulate actin dynamics withingrowth cones and to control neurite outgrowth during devel-opment (Bagrodia and Cerione 1999). Rac appears to be acentral player in the regulation of neurite outgrowth andseems to be extensively regulated. It has been reported thatRhoG, the small GTPase, mediates neurite outgrowth inneuronal cells through a signaling cascade that activatesRac1 (Causeret et al. 2004). RhoG has been shown, alongwith Elmo and Dock180, to form a complex that is able toactivate Rac1. The RhoG–Elmo–Dock180 pathway appearsto be required for neurite outgrowth induced by nervegrowth factor (Katoh and Negishi 2003). Other proteins in-volved in neurite outgrowth via Rac are ALS2/Alsin. ALS2/Alsin is a recently described GEF that regulates the activ-ities of Rac but not Rho and Cdc42. ALS2 is present withinthe growth cones of neurons, where it has been shown to co-localize with Rac (Tudor et al. 2005).

Rac and cdc42 have been shown to control actin dynamicsby modulating the activity of actin-depolymerizing factor(ADF)/cofilin. Proteins of the ADF/cofilin family regulatethe actin network by increasing the off-rate of actin mono-mers from the pointed end of actin filaments and by severingfilaments (Bamburg and Wiggan 2002). ADF/cofilin activityis necessary and sufficient to transduce the effects of brain-derived neurotrophic factor on the growth-cone motility ofneurons (Gehler et al. 2004). The 2 LIM kinases, LIMK1and LIMK2, can regulate the actin-dynamizing activities ofADF/cofilin. The LIM kinases are downstream of Rho andRac GTPases (Arber et al. 1998) and, through their effectorsRho-kinase (ROCK) and p21-activated kinases (PAK), acti-vate LIMK1 and 2 (Sumi et al. 2001; Soosairajah et al.2005). Another downstream component of Rac1 that mightmodulate actin dynamics is Arp2/3. Arp2/3, an actin-bindingcomplex, plays a key role in actin nucleation (Meyer andFeldman 2002). WAVE has been known to activate the actin-nucleating Arp2/3 complex in a Rac-dependent manner(Smith and Li 2004). Src family kinases also activate N-WASP, through tyrosine phosphorylation, which inducesArp2/3-complex-mediated actin polymerization (Suetsugu etal. 2002). Cdc42/Rac can communicate with the cytoskeletonby binding the Cdc42c42/Rac interactive binding (CRIB) mo-tif, located in the GTPase-binding domain of its autoinhibitedeffector, the Wiskott–Aldrich syndrome protein (WASP)(Higgs and Pollard 2001). Activated WASP activates theArp2/3 complex to generate cellular structures, such as fi-lopodia and lamellipodia (Leung and Rosen 2005).

The direct linkage of Go, Arp2/3, and ADF/cofilin has notbeen examined; however, Go-induced activation of the Src

Fig. 2. The signaling pathways emanating from Gai/o. A connec-tions map of the signaling pathways that can be activated by the 2direct downstream effectors of Gao: RapGAPII and GRIN.Although this map shows only Gao, current data indicate that thesepathways are also operative for Gai. The connections map illus-trates pathways that have been defined in detail (e.g., Gao to Src toStat3) and others (e.g., GRIN to PAK and Rap to PAK) where,based on known binary interactions, it is reasonable to presumesuch pathways. CBR1, CB1 cannabinoid receptor; PAK, p21-acti-vated kinases; WASP, Wiskott–Aldrich syndrome protein; JNK, c-Jun N-terminal kinase; Tyr, tyrosine; Stat3, signal transducer andactivator of transcrirtion; Ser, serine.

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and Cdc42/Rac pathways could lead to the activation ofArp2/3 and ADF/cofilin, thereby regulating actin nucleationand polymerization, the basic elements controlling neuriteoutgrowth. This network is summarized in the Fig. 2.

Role of the Src/Stat3 pathwayOur study shows that Gai/o-coupled receptors activate Src

in Neuro2A cells through Rap1 and Ral. Furthermore, Srcplays a key role in CB1 receptor/Gai/o-mediated neurite out-growth; overexpression of a dominant negative of Src orCSK has been found to inhibit receptor-induced neurite out-growth (He et al. 2005).

Src tyrosine kinases have been implicated in several as-pects of neuronal development and nervous system function.However, their mechanisms of action have not been clari-fied. It has been shown that p190RhoGAP functions as adownstream substrate for Src in neuronal cells (Brouns et al.2001), but the downstream components, including the tran-scription factors in this pathway, are not fully understood.Src also appears to mediate epidermal-growth-factor-inducedneurite outgrowth, independent of mitogen-activated proteinkinase (MAPK)1,2 phosphorylation (Yang et al. 2002).

Interestingly, our studies indicate a crucial role for Stat3in mediating signals from CB1 receptors in Neuro2A cells.We found that the CB1 receptor stimulates both the tyrosineand serine/threonine phosphorylation of Stat3. It is likelythat Src mediates the tyrosine phosphorylation of Stat3;Rac1/JNK might be involved in the serine/threonine phos-phorylation. We found that Stat3 is a key signaling moleculemediating CB1-receptor/Gai/o-induced neurite outgrowth(He et al. 2005). Thus, the Src and JNK signals might con-verge at the level of Stat3. This is summarized in Fig. 2.The genes induced by Stat3 that are responsible for neuriteoutgrowth have not yet been identified; this will be a neces-sary step in understanding how Stat3-mediated gene expres-sion contributes to neurite outgrowth.

The evidence of a critical role for Stat3 in neurite outgrowthis not conclusive. It is not clear whether Stat3-mediatedgene expression is a general requirement for neurite out-growth. Activation of the Stat3 signaling pathway, butnot the Ras/MAPK1,2-dependent pathway, is required forinterleukin-6-induced neurite outgrowth in PC12 cells(Wu and Bradshaw 2000). However, one study reportedthat Stat3 activation has a negative regulatory effect onnerve-growth-factor-stimulated neurite outgrowth (Ihara etal. 1997). In a conditional gene ablation model of Stat3,motor neuron survival is significantly reduced after facialnerve lesion in an adult, indicating a role for Stat3 inneuronal survival after injury (Schweizer et al. 2002).The molecular mechanisms underlying these differenceshave not been characterized. Future studies are neededto delineate the specific upstream signals that use Stat3-mediated transcription to induce neurite outgrowth andthe genes regulated by Stat3 that are responsible forneurite outgrowth. In vivo study using Stat3 knockoutmice would be another approach to address the role ofStat3 in neurite outgrowth.

Role of MAP kinasesMost studies that use PC12 cells to identify pathways regu-

lating neuronal process formation have focused on the Ras/

MAPK1,2 pathway. It is well-established that the activation ofthe MAPK cascade can induce neurite outgrowth (Xiao and Liu2003; Waetzig and Herdegen 2005). Because Gao activatesRap1 and Rap1 is known to activate B-Raf/MAPK1,2, we ex-amined whether the activation of the cannabinoid CB1 receptorcauses MAPK1,2 activation. We found that CB1-receptor stim-ulation activated all 3 classes of MAPK: MAPK1,2; p38; andJNK. However, our studies did not show any inhibitory effectsof PD098095 on cannabinoid CB1-receptor-induced neuriteoutgrowth, indicating that, in Neuro2A cells, the MAPK1,2pathway might not be involved. We found that SB202191, ablocker of p38 and JNK, significantly inhibited receptor-stimulated neurite outgrowth. We also confirmed that Rac-JNK, rather than MAPK1,2, plays an important role in CB1receptor/Gai/o-induced neurite outgrowth, by activating theStat3 pathway (He et al. 2005).

Role of GRINGRIN is a membrane-bound protein that has been found

to directly interact with activated Gao in vitro and in vivo(Jordan et al. 1999; Chen et al. 1999). GRIN1 colocalizeswith Gao at the growth cone of neuronal cells, and promotesneurite extension in Neuro2A cells when coexpressed withconstitutively active mutant Gao. A recent study suggeststhat the binding of activated Gao to GRIN1 induces the acti-vation of Cdc42, which leads to morphological changes inneuronal cells (Nakata and Kozasa 2005). The exact role ofGRIN in Go/i-induced neurite outgrowth requires furtherstudies.

Role of other Gai/o-coupled receptors in neuriteoutgrowth

Serotonin 5-HT1A receptors are also Gai/o-coupled recep-tors, and are implicated in anxiety and depression. A recentstudy examined the effect of receptor activation on neuronalsurvival and neurite outgrowth, using Neuro2A cells trans-fected with 5-HT1A receptors and SK-N-SH cells, which endo-genously express the receptor (Fricker et al. 2005). Receptoractivation has been found to lead to increased neurite out-growth, which can be blocked by a 5-HT1A-receptor-selectiveantagonist. Neurite outgrowth can also be modulated by treat-ment with pertussis toxin or lactacystin, implicating inhibitoryG proteins and proteasomal degradation in this process. Inter-estingly, the small G protein Rap and the transcription factorStat3 are also involved; reducing the levels of Rap1 (usingsmall interfering RNA) or Stat3 (using dominant negativeStat3) significantly blocks 5-HT1A-receptor-mediated neuriteoutgrowth. Furthermore, the prolonged activation of endoge-nous 5-HT1A receptors leads to increased cell survival, evenunder starving conditions; this is completely blocked by co-treatment with the antagonist.

Physiological implicationsThe reconstruction of neuronal networks in the brain is

necessary for the treatment of neurodegenerative diseases.Injury to neurons results in a sequence of molecular and cel-lular responses that are associated with, and that might playan important role in, the mounting of a successful regenera-tive response and the ensuing recovery of function. In theinjured neuron, the rapid arrival of signals that contribute to

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cellular injury and stress is followed by the induction oftranscription factors, adhesion molecules, growth-associatedproteins, and structural components needed for axonal elon-gation (Makwana and Raivich 2005).

Peripheral neuronal cells contain numerous growth-inducingand trophic molecules, including the ciliary neurotrophic factor(CNTF), neurotrophin-3, and fibroblast growth factors (Ecken-stein et al. 1991; Funakoshi et al. 1993), which are secretedafter injury. Stat3 is the downstream target of the activatedCNTF receptor. Both CNTF and Stat3 have regulatory roles.Neuronal deletion of Stat3 results in enhanced post-traumaticcell death, implicating a neurotrophic-like role for Stat3(Schweizer et al. 2002). Deletion of CNTF, which is abun-dantly present in myelinated Schwann cells but not in or aroundthe cell bodies of axotomized motoneurons (Rende et al. 1992;Dobrea et al. 1992), causes a delay in the appearance of thephosphorylated Stat3 and its nuclear translocation in neuronalcell bodies. This indicates a signaling cascade from the local re-lease of CNTF by damaged myelinated Schwann cells, the localaction of CNTF on adjacent axons, intra-axonal phosphoryla-tion of Stat3, to its retrograde transport to the cell bodies of in-jured neurons.

Inhibition of Ras, Raf, and the MAPK1,2, molecules thatare upregulated after axonal injury (Kiryu et al. 1995) havebeen shown to inhibit the survival and neurite outgrowth ofembryonic neurons (Ihara et al. 1997; Borasio et al. 1989).Conversely, studies of adult neurons suggest a loss or re-versal of this prosurvival/proneurite outgrowth role (Liuand Snider 2001; Wiklund et al. 2002), stressing the dis-similarity in the embryonic/neonatal and adult signaling.After activation of the MAPK pathways, phosphorylationand nuclear localization of a host of transcription factors,including c-jun, junD, ATF3, P311, Sox11, and Stat3, con-tribute to the change in gene expression of the injured neu-ron. Neuronal-specific deletion of Stat3 increases neuronalcell death after injury (Schweizer et al. 2002), to a degreesimilar to that observed for CNTF and leukemia inhibitoryfactor (Sendtner et al. 1996), pointing to the role of Stat3as an intracellular-survival-promoting factor.

Consistent with the above reports, our study suggeststhat Stat3, instead of MAPK, is the central molecule me-diating CB1-receptor/Gai/o-mediated neurite outgrowth.CB1-receptor/Gai/o-induced Src activation is the key stepleading to Stat3 activation (by tyrosine phosphorylation),whereas activation of Rac/JNK further enhances the activ-ity of Stat3 (He et al. 2005). The physiologic role of theCB1 receptor and its downstream signaling pathways inneurite outgrowth and neurite regeneration need to bestudied further in vitro and in vivo.

PerspectiveCB1 receptor/Gai/o regulates neurite outgrowth in Neuro2A

cells through a complex signaling network, containing a cas-cade of small GTPases (Rap1, Ral, and Rac, and protein kin-ases Src and JNK), which converge to activate Stat3. Recentstudies, including ours, provide evidence that multiple signal-ing pathways downstream of Gai/o are involved in regulatingneurite outgrowth. Our study also suggests that Stat3 plays akey role in integrating upstream signaling with gene expres-sion in regulating the process of neurite outgrowth down-

stream of CB1 receptor/Gai/o. Further study is required toaddress the role of Stat3 in signal-mediated neuronal regener-ation in vivo.

AcknowledgementsThis work was supported by National Institutes of Health

grants DK-65495 (to J.H.), GM-54508 and CA-81050 (toR.I.), and to an individual NRSA fellowship F31GM65065(to S.R.N.).

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