Mini-Review

Neurotensin and the NeurotensinReceptor-3 in Microglial Cells

Stephane Martin, Eleni Dicou, Jean-Pierre Vincent, and Jean Mazella*

Institut de Pharmacologie Moleculaire et Cellulaire, Unite Mixte de Recherche 6097 du Centre Nationalde la Recherche Scientifique, Sophia Antipolis, Valbonne, France

Microglia motility plays a crucial role in response tolesion or exocytotoxic damage of the cerebral tissue.The neuropeptide neurotensin elicited the migration ofthe human microglial cell line C13NJ by a mechanismdependent on both phosphatidylinositol-3 kinase (PI3kinase) and mitogen-activated protein (MAP) kinasespathways. The effect of neurotensin on cell migrationwas blocked by the neurotensin receptor-3 propeptide,a selective ligand of this receptor. The type I neuroten-sin receptor-3 was the only known neurotensin receptorexpressed in these microglial cells, and its activation ledto the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, theeffect of neurotensin on cell migration was preceded bya profound modification of the F-actin cytoskeleton,particularly by the rapid formation of numerous cell filo-podia. Both the motility and the filopodia appearanceinduced by neurotensin were totally blocked by selec-tive inhibitors of MAP kinases or PI3 kinase pathways.In the murine microglial cell line N11, the neurotensinreceptor-3 is also the only neurotensin receptorexpressed, and its activation by neurotensin leads tothe phosphorylation of both Erk1/2 and Akt. In thesecells, neurotensin induces the gene expression of sev-eral cytokines/chemokines, including MIP-2, MCP-1,interleukin-1b and tumor necrosis factor-a. This induc-tion is dependent on both protein kinases pathways. Weobserved that the effect of neurotensin on the cytokine/chemokine expression is also inhibited by the neuroten-sin receptor-3 propeptide. This is the demonstrationthat the neurotensin receptor-3 is functional and medi-ates both the migratory action of neurotensin and itsinduction of chemokines/cytokines expression.VVC 2005 Wiley-Liss, Inc.

Key words: microglia; neurotensin receptor-3; cytokines/chemokines; sortilin; migration; PI3 kinase; MAP kinase

Brain microglia macrophages are considered tofunction as monocytes or tissue macrophages of thesomatic immune system (Gehrmann et al., 1995). Thesecells, when activated during a brain injury, proliferate in

situ (Marty et al., 1991) and display directed migrationin order to eliminate damaged neurons or invadingmicroorganisms (Brockhaus et al., 1996). Activation ofmicroglia can lead to the production of a series of cyto-toxic substances, including nitric oxide, proteases, andproinflammatory cytokines (Banati et al., 1993). In thecase of neurodegenerative disorders such as Alzheimer’sdisease (AD), microglial cells are considered to beresponsible for the neurotoxicity leading to the formationof neurofibrillary tangles after activation by the amyloidpeptide. However, another hypothesis for the role ofmicroglia in AD has been recently suggested. The micro-glial dysfunction would become manifest in decreasedclearance of Ab protein as well as in diminished trophicsupport and increased production of neurotoxins leading toneuronal dysfunction, the latter being accompanied by Abproduction (for review see Streit, 2004).

Several neuropeptides have been described as mod-ulating the immune responses in the peripheral system(De la Fuente et al., 1998; Delgado et al., 1999). Thus,the neuropeptide neurotensin (NT) has been reported toregulate some cellular functions of the peripheralimmune system (Koff and Dunegean, 1985; Garridoet al., 1992; De la Fuente et al., 1993), including cutane-ous inflammatory process (Ramez et al., 2001). How-ever, the mechanism of NT action and the receptor typeinvolved in the effects of the peptide on central macro-phages remain unknown. NT responses are known todate to be mediated through at least three receptors

Stephane Martin’s current address is MRC Centre for Synaptic Plasticity,

Department of Anatomy, School of Medical Sciences, Bristol University,

Bristol, BS8 1TD United Kingdom.

*Correspondence to: Jean Mazella, Institut de Pharmacologie Moleculaire

et Cellulaire, Unite Mixte de Recherche 6097 du Centre National de la

Recherche Scientifique, 660 route des lucioles, Sophia Antipolis, 06560

Valbonne, France. E-mail: mazella@ipmc.cnrs.fr

Received 14 September 2004; Accepted 23 September 2004

Published online 14 June 2005 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.20477

Contract grant sponsor: Centre National de la Recherche Scientifique

(CNRS); Contract grant number: PICS 2051.

Journal of Neuroscience Research 81:322–326 (2005)

' 2005 Wiley-Liss, Inc.

identified (for review see Vincent et al., 1999). The firsttwo NT receptors (NTR1 and NTR2) belong to thefamily of G-protein-coupled receptors (GPCRs; Tanakaet al., 1990; Chalon et al., 1996; Mazella et al., 1996).The third NT receptor, NTR3 (Mazella et al., 1998),also called sortilin (Petersen et al., 1997), is a type Ireceptor belonging to the new family of receptors shar-ing an N-terminal luminal domain related to the yeastsorting receptor Vps10p (Marcusson et al., 1994). Therole of the two GPCRs NTR1 and NTR2 in the effectsof NT has been partially elucidated by using the NTantagonist SR48692, which is selective for the NTR1(Betancur et al., 1997; Vincent et al., 1999). The NTR1is functionally coupled to phospholipase C (Hermanset al., 1992; Chabry et al., 1994); it induces MAP kinasephosphorylation (Poinot-Chazel et al., 1996) and isresponsible for the NT-induced modulation of dopami-nergic transmission (Nemeroff, 1986). The NTR2, forwhich the cellular coupling remains to be clarified,seems to be involved in the analgesic effect of the pep-tide (Dubuc et al., 1999). The role of the recentlycloned NTR3 as an NT receptor remains speculative,insofar as this protein has been shown to bind severalother ligands, such as the receptor-associated protein(Petersen et al., 1997), lipoprotein lipase (Nielsen et al.,1999), and more recently to the precursor of nervegrowth factor (Nykjaer et al., 2004). However, this NTbinding protein is the only one that is present in all thecancer cell lines on which NT exerts a proliferativeeffect (Dal Farra et al., 2001). When stably expressedinto CHO cells, this receptor responds to NT in thethymidine incorporation assay (Dal Farra et al., 2001)and is maintained at the cell surface, although it effi-ciently internalizes the peptide (Navarro et al., 2001).

In this study, we looked at the role of NT in cell sig-naling, migration, and expression of cytokines/chemokinesof microglial cell lines from human and mouse origins.These immortalized cells possess the macrophagic charac-teristics of adherence and phagocytosis and express severalmacrophagic antigens (Righi et al., 1989; Janabi et al.,1995). We found that only the NTR3 was expressed inthese cells and that NT fullfills the function of a modula-tor/activator of brain macrophages. We also demonstratedthat the NTR3 propeptide, released from the precursorform of the receptor (Munck Petersen et al., 1999), is apotent inhibitor of the NT-induced cell activation.

MICROGLIAL CELLS EXPRESS THE NEURO-TENSIN RECEPTOR-3/SORTILIN

By performing RT-PCR experiments with specificoligodeoxynucleotides for each of the three known NTreceptors, we observed that only the NTR3 mRNA isdetected in both the human (C13NJ) and the mouse(N11) microglial cell lines. The expression was con-firmed by Western blot analysis and also by photoaffinitycross-linking: with a photoreactive NT analogue, only a100-kDa protein can be specifically labeled at the micro-glial cell surface (Martin et al., 2003; Dicou et al., 2004).The affinity of NT for these cell surface binding sites is

2.5 6 0.48 nM and 4 6 1.5 nM in human and mousemicroglial cells, respectively.

In microglial cells, the subcellular location of theNTR3 was assessed by fluorescent immunocytochemis-try and confocal imaging. As observed in other cellsexpressing this receptor (Petersen et al., 1997; Morinvilleet al., 2004), the bulk (about 80–90%) of NTR3 labelingwas intracellular, concentrated in the TGN compartment(Fig. 1). However, a less intense labeling (10–20%) wasalso observed on the cell surface and on cellular neurite-like processes (Fig. 1).

FUNCTIONAL SIGNALING OF THE NTR3IN MICROGLIAL CELLS

NT rapidly and transiently stimulated the phos-phorylation of mitogen-activated protein (MAP) kinasesand extracellular signaling-regulated kinases (ERK1/2) inboth C13NJ and N11 cells. The stimulatory effect wasmaximal after 2.5–3 min, and the basal level was recov-ered after 10 min (Fig. 2A). Standardization of the sameblots with either the anti-NTR3 or the total anti-ERK1/2 antibodies indicated a maximal fourfold stimu-lation of phospho-ERK at 2.5–3 min in both cells (Fig.2A). The influence of NT on the phosphatidylinositol-3(PI3) kinase pathway was also investigated by measuringthe phosphorylation of Akt. NT also rapidly activatedAkt (3–5 min), and this activation was more long-lasting,with a maximal phosphorylation level obtained at 30min in C13NJ cells and at 10 min in N11 cells (Fig.2B). The effects of NT on the phosphorylation ofERK1/2 and Akt were prevented by the correspondingspecific inhibitors PD98059 and wortmannin andLY294002, respectively.

Fig. 1. Immunofluorescent labeling of NTR3 in C13NJ cells. Cellswere fixed and incubated with the rabbit polyclonal anti-NTR3,then with a Texas red-conjugated anti-rabbit antibody, before analy-sis by confocal microscopy. Arrowheads show that the NTR3 isessentially localized in the trans-Golgi compartment but is alsoexpressed at the cell surface.

Functional Neurotensin Receptor-3 in Microglia 323

BIOLOGICAL RESPONSES

NT did not stimulate microglial cell proliferationbut efficiently activated human C13NJ migration, asdemonstrated by using both a wound-healing model anda chemotaxis assay (Martin et al., 2003). The migratoryeffect is mediated by a mechanism dependent on bothPI3 kinase and MAP kinase pathways. Interestingly, theNT-induced C13NJ migration was shown to be pre-ceded by a profound modification of the F-actin cytos-keleton, particularly by the rapid formation of numerouscell filopodia, that is also blocked by the PI3 and MAPkinase inhibitors. This effect strengthens the putative roleof NT in microglia activation, in that, in response toinjury or inflammation, microglial cells can activelymigrate toward the damaged region of the brain throughseveral factors involving receptors for motility.

In the mouse N11 cell line, NT was shown to bea potent inducer of gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin (IL)-1b and tumor necrosis factor (TNF)-a. This inductionis dependent on both PI3 kinase and MAP kinases path-ways. As an example, the increase in expression of theMCP-1 mRNA was correlated with an increase in theamount of released protein (Fig. 3). Because cytokines/chemokines have been shown to be potent chemoattrac-tants, activation of their synthesis by NT can lead to cellmigration, as observed in C13NJ cells.

THE NTR3 PROPEPTIDE ANTAGONIZESNT EFFECTS

The 44-amino-acid peptide released from the furinmaturation of the precursor form of the NTR3/sortilinhas been demonstrated to bind the receptor (MunckPetersen et al., 1999). On C13NJ cells, the propeptide

binds to the NTR3 with an affinity close to that of NT(8 nM). Both in human and in mouse microgila cells,the propeptide acts as an antagonist of NT on theNTR3. Indeed, the propeptide not only dose depend-ently abolished the migration effect of NT on C13NJcells but also totally reversed NT activation of geneexpression of cytokines/chemokines in N11 cells.

CONCLUSIONS AND PERSPECTIVES

The expression of NTR3 alone in microglial cellsis of special interest, because this receptor coexists in

Fig. 3. NT stimulation of MCP-1 mRNA and protein expression inN11 cells. mRNA expression was measured by RT-PCR experi-ments from cells incubated for various times with 0.1 mM NT at378C. The amount of released MCP-1 was measured from superna-tants of the same NT-treated cells by ELISA. Histograms representmean values 6 SEM from six independent experiments for RT-PCR values and from four determinations from two independentexperiments for the protein values.

Fig. 2. Effect of NT on the phosphorylation of MAP kinases ERK1/2 (A) and of Akt (B) in C13NJ andN11 cells. Human (open circles) and mouse (solid circles) microglial cells were stimulated with 10 nMNT for various times. The phosphorylation of MAP kinases ERK1/2 and of Akt was determined byimmunoblotting with antibodies directed against their corresponding phosphorylated active forms. Datawere standardized from three different experiments by using the labeling obtained on the same blots withthe total ERK and Akt antibodies and are expressed as mean 6 SEM.

324 Martin et al.

various cells and tissues with at least another GPCR NTreceptor, making it difficult to identify the NT receptorresponsible for an observed biological or cellular effect ofthe peptide. The fact that the NTR3 is the only NTreceptor endogenously expressed both in human and inmouse microglia allowed us to establish for the first timeits functional role. NTR3 is specifically involved notonly in the NT-induced microglial cell migration butalso in the effect of the peptide on the gene expressionand release of several cytokines/chemokines from micro-glia. However, the intracellular tail of the NTR3 isprobably too short to allow this protein to transduce acellular signal by itself. As demonstrated for the role ofNTR3 in the pro-NGF-induced apoptosis in neurons(Nykjaer et al., 2004) and in the NT-elicited signalingin HT29 cells (Martin et al., 2002), the functional roleof NTR3 in microglial cells probably necessitates itsassociation with a glial protein partner that remains to beidentified.

The actions of NT on brain macrophages suggest arole for the neuropeptide in the recruitment and theactivation of these cells and in the proinflammatoryprocess that may contribute to the pathophysiology ofcerebral degenerative diseases, such as Parkinson’s andAlzheimer’s diseases. The important finding is that boththe NT-induced microglial cell migration and the NTactivation of cytokines/chemokines expression are effi-ciently blocked by the NTR3 propeptide. Further stud-ies investigating whether activation of microglial cellsby NT leads to degeneration of neuronal cells in cocul-ture systems would help to determine the potential useof propeptide analogues as protectors against inflamma-tion and neurotoxicity. This could lead to the develop-ment of new therapeutic strategies for the treatment ofneuroinflammation observed in severe cerebral braindamage.

ACKNOWLEDGMENTS

We thank Dr. Claus Pertersen and Dr. Morten S.Nielsen (Aarhus, Denmark) for the generous gift ofNTR3 antibodies.

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