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Neurobiology of Aging 31 (2010) 118–128

A novel anti-inflammatory role of NCAM-derived mimetic peptide, FGL

Eric J. Downer a, Thelma R. Cowley a, Anthony Lyons a, Kingston H.G. Mills b,Vladimir Berezin c, Elisabeth Bock c, Marina A. Lynch a,∗

a Trinity College Institute for Neuroscience, Physiology Department, Trinity College, Dublin 2, Irelandb Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland

c Protein Laboratory, Institute of Neuroscience and Pharmacology, School of Medicine,University of Copenhagen, Copenhagen, Denmark

Received 4 December 2007; received in revised form 3 March 2008; accepted 20 March 2008Available online 12 May 2008

bstract

Age-related cognitive deficits in hippocampus are correlated with neuroinflammatory changes, typified by increased pro-inflammatoryytokine production and microglial activation. We provide evidence that the neural cell adhesion molecule (NCAM)-derived mimetic peptide,G loop (FGL), acts as a novel anti-inflammatory agent. Administration of FGL to aged rats attenuated the increased expression of markers ofctivated microglia, the increase in pro-inflammatory interleukin-1� (IL-1�) and the impairment in long-term potentiation (LTP). We reporthat the age-related increase in microglial activation was accompanied by decreased expression of neuronal CD200, and suggest that theroclivity of FGL to suppress microglial activation is due to its stimulatory effect on neuronal CD200. We demonstrate that FGL enhancednterleukin-4 (IL-4) release from glial cells and IL-4 in turn enhanced neuronal CD200 in vitro. We provide evidence that the increase in

D200 is reliant on IL-4-induced extracellular signal-regulated kinase (ERK) signal transduction. These findings provide the first evidencef a role for FGL as an anti-inflammatory agent and identify a mechanism by which FGL controls microglial activation.

2008 Elsevier Inc. All rights reserved.

eywords: Age; Hippocampus; NCAM; Microglia; FGL; CD200; Interleukin-1� (IL-1�); Interleukin-4 (IL-4); Extracellular signal-regulated kinase (ERK);

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ong-term potentiation (LTP)

. Introduction

Several groups have correlated aging with cognitive dys-unction (Barnes, 1988; Gemma and Bickford, 2007). Inddition to cognitive decline, brain aging is associated witheficits in long-term potentiation (LTP) in perforant path-ranule cell synapses (Murray and Lynch, 1998; McGahont al., 1999), reactive oxygen species (ROS) accumula-ion (O’Donnell et al., 2000), neuronal (Hwang et al.,006; Potier et al., 2006) and synaptic (Geinisman et al.,992a,b) loss. Importantly these are accompanied by inflam-atory changes within the hippocampus, characterised by

n alteration in pro- and anti-inflammatory cytokine bal-nce, down-regulation of neuronal CD200 expression andubsequent microglial cell activation (Ogura et al., 1994;

∗ Corresponding author. Tel.: +353 1 896 8531; fax: +353 1 679 3545.E-mail address: lynchma@tcd.ie (M.A. Lynch).

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197-4580/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.neurobiolaging.2008.03.017

riffin et al., 2006; Lyons et al., 2007a). Indeed, anndirect correlation between activation of microglia andxpression of CD200 on neurons has recently been iden-ified in hippocampus of aged animals, with evidence thateuronal CD200 expression is directly regulated by the anti-nflammatory cytokine interleukin-4 (IL-4) (Lyons et al.,007a). Furthermore, previous data has indicated that thege-related increase in hippocampal concentration of thero-inflammatory cytokine interleukin-1� (IL-1�) directlyontributes to the deficits in LTP (Lynch and Lynch, 2002).ignificantly, administration of eicosapentaenoic acid (Lyncht al., 2007) and rosiglitazone (Loane et al., 2007), which pos-ess anti-inflammatory properties, restores the age-relatedecrease in LTP. These agents also reverse the age-related

ncrease in microglial cell activation and subsequent IL-1�roduction in the hippocampus, highlighting the impor-ant role of microglial activation in attenuating synapticunction.

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E.J. Downer et al. / Neurobi

The neural cell adhesion molecule (NCAM) is expressedbundantly throughout the nervous system, where it actss a mediator of cell–cell adhesion and a trigger of sig-aling pathways (Schachner, 1997; Soroka et al., 2002).

role for NCAM in cognitive processes has been veri-ed by studies in NCAM−/− mice (Cremer et al., 1994),hile the evidence supporting its role in plasticity, learn-

ng and synaptogenesis is unequivocal (Luthl et al., 1994;elzl and Stork, 2003). NCAM modulates many signal

ransduction cascades by direct interaction with the fibrob-ast growth factor receptor (FGFR) (Kiselyov et al., 2003),eading to activation of protein kinase C (Kolkova et al.,000), phosphatidylinositol-3-kinase (PI3K) (Ditlevsen etl., 2003; Neiiendam et al., 2004) and mitogen-activatedrotein kinases (MAPKs) (Povlsen et al., 2003; Neiiendamt al., 2004). Several synthetic NCAM peptide mimeticsave been developed (Berezin and Bock, 2004), and oneuch peptide, identified as FG loop (FGL), mimics the het-rophilic interaction between NCAM and FGFR1. Indeed,GL induces a two-fold increase in FGFR1 phosphoryla-

ion (Kiselyov et al., 2003). The FGL peptide is a 15 aminocid sequence in the second F3 module of NCAM, cor-esponding to residues E681-A695 of the FG loop regionKiselyov et al., 2003). It has recently been shown thatGL acts as a neuroprotective agent in vitro and in vivoCambon et al., 2004; Neiiendam et al., 2004), and cannduce neurite outgrowth in vivo, in addition to enhancingynaptogenesis and presynaptic function in primary neu-ons (Cambon et al., 2004; Neiiendam et al., 2004). In vivodministration of FGL induces a long-lasting facilitation oferformance in hippocampus-dependent tasks (Cambon etl., 2004) and can abrogate the cognitive impairment inducedy amyloid-� (A�)25–35 administration (Klementiev et al.,007). Preclinical studies with FGL have demonstrated thebsence of systemic toxicity in rats, dogs and monkeys, withdose related pharmacokinetic profile determined in humans

Anand et al., 2007).We set out to determine if FGL modulates the neuroinflam-

ation in the hippocampus that accompanies brain aging, ando identify the mechanism by which FGL regulates microglialctivation both in vivo and in vitro. The data indicate thatreatment of aged rats with FGL attenuates microglial acti-ation, that this is due to a stimulatory effect of FGL onD200, and is associated with restoration of LTP. We show

hat FGL induces IL-4 release from glial cells in vitro, andhat IL-4 in turn regulates neuronal CD200 via extracellularignal-regulated kinase (ERK) signaling.

. Methods

.1. Animals

Male Wistar rats (Trinity College, Dublin, Ireland) aged 4onths (250–350 g) or 22 months (550–650 g) were housed

nder a 12 h light schedule with controlled ambient tempera-

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f Aging 31 (2010) 118–128 119

ure (22–23 ◦C) and maintained under veterinary supervisionhroughout the study. These experiments were performednder a license issued by the Department of Health (Ireland)nd in accordance with the guidelines laid down by the localthical committee.

.2. Animal treatments and induction of LTP

FGL (8 mg/kg) or vehicle (sterile water) was injected sub-utaneously on alternate days for 3 weeks. The dose and routef administration was based on previous publications (Sechert al., 2006). The injected form (dimeric) of the peptide con-ists of two FGL monomers linked at the N-terminal. Thisimeric form of FGL has been selected for clinical devel-pment (Anand et al., 2007). Rats were anaesthetized 24 hollowing final FGL injection by intraperitoneal injection ofrethane (1.5 g/kg) and the ability of rats to sustain LTP in per-orant path-granule cell synapses was assessed as previouslyescribed (Nolan et al., 2005). Following LTP experiments,ats were killed by cervical dislocation and hippocampal tis-ue frozen.

.3. Preparation of primary cultures and treatments

Mixed glia were prepared from the cortices of 1-day-ld Wistar rats, C57/BL6 (wildtype) mice and IL-4−/−ice back crossed onto C57/BL6 mice (Trinity College,ublin, Ireland), and plated (0.25 × 106 cells/ml) as previ-usly described (Nolan et al., 2005). After 2 weeks, mixedlia were pre-treated with FGL (0.1–100 �g/ml) for 24 hnd incubated in the presence or absence of lipopolysac-haride (LPS) (1 �g/ml; 3000 endotoxin units/ml; Sigma,K) for 24 h. For preparation of microglia and astro-

ytes, dissected cortices were chopped, added to Dulbecco’sodified Eagle’s medium (DMEM) (Invitrogen, Ireland),

riturated, passed through a sterile mesh filter (40 �m) andentrifuged (2000 × g for 3 min at 20 ◦C). The pellet wasesuspended in DMEM and plated onto T25 flasks. Cellsere grown at 37 ◦C in a humidified environment (5%O2:95% air). DMEM containing macrophage colony stim-lating factor (M-CSF) (20 ng/ml; R&D Systems, UK) andranulocyte macrophage colony stimulating factor (GM-SF) (50 �g/ml; R&D Systems, UK) was changed after 1,and 8 days. At day 14, non-adherent cells (microglia)ere isolated by shaking, centrifuged (2000 × g for 5 min

t 20 ◦C), and the microglia-enriched pellet was resuspendedn DMEM. Microglia were plated (1 × 105 cells/ml) on 10-

m diameter coverslips coated with poly-l-lysine (Sigma,K).Primary astrocytes were isolated from mixed glia at day

4 by removing non-adherent cells with mechanical shak-ng and harvesting by trypsinization (0.25% Trypsin-0.02%

DTA). Cells were centrifuged (2000 × g for 5 min at 20 ◦C)nd the astrocyte-enriched pellet was resuspended in DMEM.strocytes were plated (1 × 105 cells/ml) on 6-well plates.fter 1 day, astrocytes and microglia were pre-treated with

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GL (1–100 �g/ml) for 24 h and incubated in the presence orbsence of LPS (1 �g/ml; Sigma, UK) for 24 h.

Cultured neurons were prepared from the cortices of-day-old Wistar rats (BioResources Unit, Trinity Col-ege, Dublin) and plated (0.25 × 106 cells/ml) as previouslyescribed (Nolan et al., 2004). Neurons were pre-treated withhe MEK inhibitor PD98059 (50 �M; Merck Biosciencestd., UK) for 1 h prior to exposure to IL-4 (200 ng/ml; R&Dystems, UK) for 20 h.

.4. Immunostaining for major histocompatibilityomplex II (MHCII)

Mixed glial cells were fixed in ice-cold ethanol andssessed for MHCII expression as previously describedLyons et al., 2007a).

.5. Analysis of IL-1β and IL-4

IL-1� and IL-4 were assessed in samples of supernatantrom glial cultures and in hippocampal homogenates bynzyme-linked immunosorbent assay (R&D Systems, UK)s previously described (Nolan et al., 2005).

.6. RNA isolation and cDNA synthesis

RNA was extracted from hippocampal tissue using aucleoSpin® RNAII isolation kit (Macherey-Nagel Inc.,ermany) and the concentration was determined using aV–vis spectrophotometer (Beckman Coulter Inc., Ireland).

DNA synthesis was performed on 1–2 �g RNA using a Highapacity cDNA RT Kit (Applied Biosystems, USA).

.7. Real-time PCR

Real-time PCR primers were delivered as “Taqman®

ene Expression Assays” containing forward and reverserimers, and a FAM-labeled MGB Taqman probe for eachene (Applied Biosystems, USA). Primers used were as fol-ows: IL-1� and CD200 (Taqman® Gene Expression Assayos. Rn00580432 m1 and Rn00580478 m1, respectively). Ain 4 dilution of cDNA was prepared and real-time PCR

erformed using Applied Biosystems 7300 Real-time PCRystem. cDNA was mixed with qPCRTM Mastermix PlusApplied Biosystems, USA) and the respective gene assay.at �-actin was used as an endogenous control and expres-

ion was conducted using a gene expression assay containingorward and reverse primers and a VIC-labeled MGB Taq-an probe (#4352340E; Applied Biosystems, USA). Forty

ycles were run as follows: 10 min at 95 ◦C and for each cycle,5 s at 95 ◦C and 1 min at 60 ◦C. Gene expression was cal-ulated relative to the endogenous control and analysis waserformed using the 2−��CT method.

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f Aging 31 (2010) 118–128

.8. Western immunoblotting

CD200, ERK, intercellular adhesion molecule-1 (ICAM-) and CD86 were separated by SDS-PAGE as previouslyescribed (Lyons et al., 2007a). Protein expression wasetected using goat polyclonal CD200 antibody (1:200;vernight at 4 ◦C), goat polyclonal CD86 antibody (1:200;vernight at 4 ◦C), mouse monoclonal ICAM-1 antibody1:200; 2 h at room temperature) and mouse monoclonalnti-phospho-ERK antibody (1:3000; 2 h at room tempera-ure). All primary antibodies were obtained from Santa Cruziotechnology Inc., USA. Membranes were stripped (Re-lot Plus; Chemicon International Inc., CA) and incubatedith mouse monoclonal anti-�-actin antibody (1:10,000;vernight at 4 ◦C, Sigma). For analysis of ERK, mem-ranes were incubated with mouse monoclonal anti-ERK1:2000; 2 h at room temperature, Santa Cruz Biotechnologync., USA). Molecular weight markers were used to con-rm molecular weight of bands which were quantified byensitometric analysis. Data are expressed as densitometricnits representing the ratio of density of the target protein to-actin. No significant changes were observed in �-actin.

.9. Statistical analysis

Data were analysed using Student’s t-test for indepen-ent means (data in Fig. 4B), one-way or two-way analysisf variance (ANOVA) as appropriate. When analysis byNOVA indicated significance (p < 0.05), the post hoc Stu-ent Newman–Keuls test was used. In Fig. 3C, a mixedactorial analysis with repeated measures was performed.

here correlations are performed the Pearson’s r value iseported. Data are expressed as means with standard errorsS.E.).

. Results

Much evidence has indicated that the neuronal membranessociated glycoprotein, CD200, is pivotal in regulating thectivation state of microglia (Hoek et al., 2000). Indeed, weave recently shown that neuronal CD200 protein expressions down-regulated (decreased 1.4 fold) in the hippocampus ofged rats, and that this is inversely correlated with expressionf markers of microglial cell activation (Lyons et al., 2007a).ere we analysed CD200 mRNA and protein expression

n hippocampal tissue prepared from vehicle- and FGL-reated, young and aged, rats. Interestingly, two-way ANOVAnalysis revealed that treatment of aged animals with FGLeversed the age-related decline in CD200 mRNA expressionage × FGL interaction: F(1,19) = 8.04, +p < 0.05; Fig. 1A),nd significantly increased CD200 protein expression in

issue prepared from aged animals (age × FGL interac-ion: F(1,25) = 34.78, ++p < 0.01; Fig. 1B). CD200 mRNA*p < 0.05; Fig. 1A) and protein (#p < 0.05; Fig. 1B) expres-ion were significantly decreased in hippocampal tissue

E.J. Downer et al. / Neurobiology of Aging 31 (2010) 118–128 121

Fig. 1. FGL-induced CD200 expression regulates microglial activation in vivo. (A) CD200 mRNA and (B) protein expression were decreased in vehicle-treatedaged rats (*p < 0.05 and #p < 0.05 versus young control group for mRNA and protein, respectively). These changes were reversed in aged rats treated withFGL (+p < 0.05 and ++p < 0.01 versus aged control group for mRNA and protein, respectively). Values are arbitrary and densitometric units from five to sevenanimals per experimental group. (B) FGL increased CD200 protein in young FGL-treated animals (*p < 0.05 versus young control group). (C) CD86 and (D)I us youna ICAMa tion betC

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CAM-1 protein were increased in vehicle-treated aged rats (*p < 0.05 versged rats (++p < 0.01 and +p < 0.05 versus aged control group for CD86 andnimals per experimental group. Values are mean ± S.E. (E) Inverse correlaD200 protein expression (p < 0.05).

repared from aged, compared with young rats, as deter-ined by post hoc analysis. Furthermore, treatment of young

nimals with FGL enhanced CD200 protein expression, asetermined by post hoc analysis (*p < 0.05; Fig. 1B). To

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g control group for both). These changes were attenuated in FGL-treated-1, respectively). Data are densitometric measurements from five to seven

ween CD86 and CD200 protein expression (p < 0.01) and (F), ICAM-1 and

irectly assess whether the decrease in CD200 was coupledith increased microglial activation, and to assess whetherGL modulated microglial activation status, we examined

he expression of two markers of microglial activation, the

122 E.J. Downer et al. / Neurobiology of Aging 31 (2010) 118–128

Fig. 2. FGL attenuates IL-1� production in vivo and in vitro. (A) IL-1� mRNA and (B), protein were increased in vehicle-treated aged rats (*p < 0.05 and**p < 0.01 versus young control group for mRNA and protein, respectively). These effects were attenuated in FGL-treated aged rats (+p < 0.05 and ++p < 0.01versus aged control group for mRNA and protein, respectively). Values are arbitrary units from five to seven animals per experimental group or the concentrationof IL-1� in pg/mg protein for seven animals per experimental group. (C) IL-1� production was increased in LPS-treated (1 �g/ml; 24 h) mixed glia (***p < 0.001versus untreated glia). This was attenuated by pre-treatment with FGL (24 h) at 1 �g/ml, 10 �g/ml and 100 �g/ml prior to LPS exposure (+p < 0.05; ++p < 0.01and +++p < 0.001, respectively, versus LPS-treated glia). Treatment with FGL (10 �g/ml; 24 h) alone had no effect on basal IL-1� levels. Values are expressed asconcentration of IL-1� in pg/ml and are representative of three independent experiments. (D) Light microscopic images of MHCII staining in (i) vehicle-treated,(ii) LPS-treated (1 �g/ml; 24 h) and (iii) FGL (10 �g/ml; 24 h pre-treatment) + LPS-treated (1 �g/ml; 24 h) mixed glia. The images are representative of threeseparate experiments. Scale bars 100 �m. (E) IL-1� was increased in LPS-treated (1 �g/ml; 24 h) microglia (***p < 0.001 versus untreated microglia). Thiswas attenuated by pre-treatment with FGL (24 h) at 10 �g/ml and 100 �g/ml prior to LPS (++p < 0.01 and +++p < 0.001 versus LPS-treated microglia). Valuesare expressed as concentration of IL-1� in pg/ml and are representative of three independent experiments. (F) IL-1� was increased in LPS-treated (1 �g/ml;2 with FGv 1� in pgm

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4 h) astrocytes (***p < 0.001 versus untreated astrocytes). Pre-treatmentersus LPS-treated astrocytes). Values are expressed as concentration of IL-ean ± S.E.

o-stimulatory molecule CD86 and the glycoprotein, ICAM-, in hippocampal tissue prepared from vehicle-treated andGL-treated, young and aged, rats. The combined age × FGL

nteraction by two-way ANOVA had a significant impactn CD86 (F(1,19) = 6.12, ++p < 0.01; Fig. 1C) and ICAM-

(F(1,19) = 6.76, +p < 0.05; Fig. 1D) protein expression.ost hoc analysis demonstrated that CD86 and ICAM-1

rotein expressions were both increased in hippocampalissue obtained from vehicle-treated aged, compared withoung, rats (*p < 0.05; Fig. 1C and D). Furthermore, postoc analysis demonstrated that FGL significantly attenuated

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L (24 h) at 100 �g/ml attenuated the LPS-induced increase (+++p < 0.001/ml and are representative of three independent experiments. All values are

he age-related increase in CD86 (++p < 0.01; Fig. 1C) andCAM-1 (+p < 0.05; Fig. 1D). This argues that FGL has theroclivity to reverse the increase in microglial activationhat accompanies aging in the rat hippocampus due to itstimulatory effect of neuronal CD200 expression. Indeed, aignificant inverse correlation between protein expression ofD200 and CD86, (r = −0.50, n = 20, p < 0.01; Fig. 1E) and

CAM-1 (r = −0.47, n = 20, p < 0.05; Fig. 1F) was observed.We considered that the previously observed age-related

ncrease in pro-inflammatory IL-1� concentration in hip-ocampus (Lynch et al., 2007) might be attenuated by

ology of Aging 31 (2010) 118–128 123

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Fig. 3. FGL reverses the age- and LPS-induced decline in IL-4 production.(A) IL-4 protein levels were decreased in vehicle-treated aged rats (*p < 0.05versus young control group). This was reversed in FGL-treated aged rats(+p < 0.05 versus aged control group). Values are expressed as concentrationof IL-4 in pg/mg protein for seven animals per experimental group. (B) IL-4protein was decreased in LPS-treated (1 �g/ml; 24 h) mixed glia (*p < 0.05versus untreated mixed glia). This was reversed by FGL pre-treatment (24 h)at concentrations of 10 �g/ml and 100 �g/ml prior to LPS exposure (1 �g/ml,24 h; +p < 0.05 and +++p < 0.001 respectively, versus LPS-treated mixed glialcells). FGL (100 �g/ml) in the presence of LPS (1 �g/ml; 24 h) enhanced IL-4 above control levels (#p < 0.05 versus untreated mixed glia). FGL treatment(10 �g/ml; 24 h) alone showed an enhancement of IL-4 (p = 0.09 versusuntreated mixed glia). Values are expressed as concentration of IL-4 in pg/mland are representative of three independent experiments. (C) Treatment ofmixed glia prepared from wildtype and IL-4−/− mice with LPS (1 �g/ml;24 h) increased IL-1� production (*p < 0.05 versus untreated mixed glialcells from wildtype mice and ***p < 0.001 versus untreated mixed glialcells from IL-4−/− mice). Pre-treatment with FGL (10 �g/ml) attenuated thisincrease in mixed glia prepared from wildtype mice (+p < 0.05 versus LPS-treated glia), but not IL-4−/− mice. Values are expressed as concentration ofIL-1� in pg/ml and are representative of two independent experiments. Allvalues are mean ± S.E.

E.J. Downer et al. / Neurobi

GL. Two-way ANOVA analysis determined that the com-ined age × FGL interaction had a significant impact onL-1� mRNA (F(1,19) = 3.84, +p < 0.05; Fig. 2A) and pro-ein (F(1,24) = 4.48, ++p < 0.01; Fig. 2B) expression. Post hocnalysis revealed that hippocampal IL-1� mRNA and proteinxpression were increased with age (*p < 0.05 and **p < 0.01or mRNA and protein; Fig. 2A and B); these changes werettenuated by FGL (+p < 0.05 and ++p < 0.01 for mRNA androtein; Fig. 2A and B).

Since it is accepted that the predominant source of IL-1� isctivated microglia (Li et al., 2003), we determined whetherGL exerted a direct effect on IL-1� release from culturedixed glia exposed to the endotoxin, LPS. One-way ANOVA

nalysis revealed that IL-1� production was significantlyncreased in LPS-treated mixed glia (***p < 0.001; Fig. 2C)nd pre-treatment with FGL (24 h) at 1 �g/ml, 10 �g/ml and00 �g/ml dose-dependently abrogated the LPS-inducedffect (+p < 0.05; ++p < 0.01; +++p < 0.001; Fig. 2C). LPSid not compromise glial cell viability as determined byhe colorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]eduction method (data not shown), but induced expressionf the marker of microglial activation, MHCII, in mixed glialultures; this was attenuated by FGL (10 �g/ml; Fig. 2D).

Considering that a mixed glial culture consists oficroglia and astrocytes, we determined whether FGL reg-

lated IL-1� release from isolated microglia and astrocytesxposed to LPS. One-way ANOVA analysis determined thatL-1� production was increased in LPS-treated microglia***p < 0.001; Fig. 2E) and FGL pre-treatment at 10 �g/mlnd 100 �g/ml attenuated the LPS-induced increase in IL-1�roduction (++p < 0.01; +++p < 0.001; Fig. 2E). In culturedstrocytes, one-way ANOVA analysis demonstrated thatL-1� production was increased in LPS-treated astrocytes***p < 0.001; Fig. 2F) and pre-treatment with FGL at00 �g/ml attenuated the LPS-induced increase in IL-1� pro-uction (+++p < 0.001; Fig. 2F). These findings suggest thatGL may exert its anti-inflammatory properties by targetingoth microglia and astrocytes, and indicate that microglialells are more responsive to the anti-inflammatory effects ofGL since lower doses of FGL abrogated LPS-induced IL-1�roduction in microglial cells compared with astrocytes.

To determine how FGL regulates CD200 expression andonsequently microglial activation, we first analysed IL-4xpression in hippocampal tissue prepared from vehicle- andGL-treated young and aged rats, and in cultured mixedlial cells exposed to LPS in the presence of FGL. Recentvidence from our laboratory has demonstrated that IL-4lays a central role in modulating expression of CD200 andicroglial activation (Lyons et al., 2007a) and a decline in

L-4 is a key neuroinflammatory change that occurs in therain with age (Maher et al., 2005; Nolan et al., 2005).

he combined age × FGL interaction by two-way ANOVAad a significant impact on IL-4 protein level (F(1,27) = 7.00,p < 0.05; Fig. 3A). In support of previous evidence, postoc analysis of the present data show that there was a sig-

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Fig. 4. FGL-induces ERK in vivo and ERK mediates IL-4-induced CD200expression. (A) FGL increased phospho-ERK expression in young and agedrats (**p < 0.01 and +++p < 0.001 versus young and aged vehicle-controlgroup, respectively). Phospho-ERK was decreased in vehicle-treated agedrats (#p < 0.05 versus vehicle-treated young rats). Values are densitomet-ric units from six to seven animals per experimental group. (B) Treatmentof cultured neurons with IL-4 (200 ng/ml; 20 h) increased phospho-ERKexpression (**p < 0.01 versus untreated cultured neurons). Values are den-sitometric units and are representative of three independent experiments. (C)Treatment of cultured neurons with IL-4 (200 ng/ml; 20 h) increased CD200expression (**p < 0.01 versus untreated neurons). Pre-treatment with theMEK inhibitor PD98059 (50 �M; 1 h) prior to IL-4 (200 ng/ml; 20 h) abro-gated this effect (++p < 0.01 versus IL-4-treated neurons). Treatment withPdA

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24 E.J. Downer et al. / Neurobi

ificant age-related decrease in IL-4 protein concentrationn hippocampal tissue prepared from aged, compared withoung, rats (Fig. 3A; *p < 0.05). FGL treatment reversed thege-related decline in IL-4 (+p < 0.05; Fig. 3A). We alsoetermined whether FGL exerted an effect on IL-4 releaserom cultured mixed glia exposed to LPS in vitro and one-ay ANOVA analysis indicated that IL-4 concentration was

ignificantly decreased in glia exposed to LPS for 24 h, com-ared with untreated glia (*p < 0.05; Fig. 3B). Pre-treatmentith FGL at concentrations of 10 �g/ml and 100 �g/ml

or 24 h significantly reversed the LPS-induced decrease inL-4 in a dose-dependent manner (+p < 0.05; +++p < 0.001;ig. 3B).

To determine the role of IL-4 in mediating the effectf FGL on IL-1� production, cultured mixed glial cellsere prepared from wildtype and IL-4−/− mice and pre-

reated with FGL (10 �g/ml) prior to LPS exposure. Fig. 3Cndicates that the combined LPS × FGL interaction by two-ay ANOVA had a significant impact on IL-1� production

rom glia prepared from wildtype mice (F(1,17) = 16.85,p < 0.05; Fig. 3C). LPS increased IL-1� release fromlia prepared from wildtype and IL-4−/− mice (*p < 0.05nd ***p < 0.001, respectively). Pre-exposure of glia fromildtype mice to FGL attenuated the LPS-induced IL-1� pro-uction (+p < 0.05; Fig. 3C); this effect was absent in cellsrepared from IL-4−/− mice. It is noteworthy that speciesifferences (rat versus mouse) in mixed glial cell response toPS were not observed, suggesting that the regulatory effectf FGL on LPS-induced cytokine production is conservedcross species.

To further investigate the mechanism by which IL-4 reg-lates neuronal CD200 we assessed the effect of FGL andL-4 on ERK signaling. IL-4 engagement of its receptoresults in ERK activation (Nelms et al., 1999) and signalingathways downstream of FGFR include the MAPK pathwayEswarakumar et al., 2005). Indeed, FGL has the procliv-ty to induce ERK phosphorylation in cerebellar granuleeurons (Neiiendam et al., 2004). Consistent with this, theombined age × FGL interaction by two-way ANOVA had aignificant impact on ERK phosphorylation (F(1,25) = 41.53,++p < 0.001; Fig. 4A), with post hoc analysis revealing anGL-induced increase in ERK phosphorylation in hippocam-al tissue obtained from young (**p < 0.01; Fig. 4A) and aged+++p < 0.001; Fig. 4A) rats. Furthermore, post hoc analy-is revealed a concomitant decline in ERK phosphorylationn the hippocampus of vehicle-treated aged, compared withoung, rats (#p < 0.05; Fig. 4A). Consistent with previousbservations (Maher et al., 2005), we demonstrate that IL-4ignificantly induced ERK phosphorylation in cultured neu-ons (**p < 0.01; Fig. 4B). Furthermore, IL-4 was showno enhance CD200 protein expression in cultured neurons**p < 0.01; Fig. 4C), while the stimulatory effect of IL-4 on

D200 was abated by pre-exposure of neurons to the MEK

nhibitor, PD98059 (++p < 0.01), indicating that the effect ofL-4 on CD200 is dependent on neuronal ERK signalingFig. 4C).

if2A

D98059 (50 �M; 20 h) alone had no effect on CD200 expression. Values areensitometric units and are representative of three independent experiments.ll values are mean ± S.E.

Much evidence indicates that maintenance of LTP isarkedly depressed in the hippocampus of aged rats

Landfield et al., 1978; Nolan et al., 2005), while further datandicates that FGL induces a long-lasting facilitation of per-

ormance in hippocampus-dependent tasks (Cambon et al.,004). The data presented here support this, with two-wayNOVA analysis of the mean changes in mean excitatory

E.J. Downer et al. / Neurobiology of Aging 31 (2010) 118–128 125

Fig. 5. FGL attenuates the age-related deficits in LTP in perforant path-granule cell synapses. (A) Tetanic stimulation (time 0) induced a sustained increasein mean population epsp slope in young vehicle- (filled symbol) and FGL-treated (open symbol) rats. (B) Responses to tetanic stimulation were attenuated invehicle-treated aged (filled symbol), but not in FGL-treated (open symbol), aged rats. (C) Data reveal a significant decrease in mean population epsp in agedr effect (v as them

pecohp(r

4

ranvairIir

mic

itiTMeiemavesriinif2aCi

ats compared with young rats (*p < 0.05); FGL significantly attenuated thisalues are derived from four consecutive responses and values are expressedean value immediately prior to tetanic stimulation).

ost-synaptic potential (epsp) slope in the last 5 min of thexperiment (compared with the mean value in the 5 min pre-eding the tetanus) revealed a significant age × FGL effectn mean epsp slope (F(1,16) = 6.62, +p < 0.05; Fig. 5C). Postoc analysis demonstrated that LTP is depressed in the hip-ocampus of aged (*p < 0.05; Fig. 5B), compared with youngFig. 5A), rats, and indicate that FGL attenuates the age-elated decrease in LTP (+p < 0.05; Fig. 5B).

. Discussion

We examined the ability of FGL to abrogate the neu-oinflammatory changes which are characteristic features ofging in the rat hippocampus. FGL induced an increase ineuronal CD200 expression and, consistent with our obser-ation that CD200 is inversely correlated with microglialctivation (Lyons et al., 2007a), reversed the age-relatedncrease in microglial activation. FGL attenuated the age-elated increase in hippocampal IL-1� and decrease inL-4, supporting the proposal that FGL acts as an anti-nflammatory agent. These changes were coupled with aestoration of LTP.

We report that FGL enhances CD200 expression, a type 1embrane glycoprotein recognized to play a role in modulat-

ng microglial activation (Barclay et al., 2002), and that thishange was accompanied by an attenuation of the age-related

auTe

+p < 0.05). Data are the means of six animals per experimental group. S.E.percent change in field epsp slope after tetanic stimulation (compared with

ncreases in CD86, ICAM and IL-1� which are characteris-ic changes associated with activation of microglia. CD200s expressed on several cell types including neurons, B and

cells and vascular endothelial cells (Preston et al., 1997;atsumoto et al., 2007), with recent evidence indicating its

xpression on glial cells (Chitnis et al., 2007). CD200 exertsts effect by binding to the CD200 receptor (CD200R), thexpression of which is restricted to myeloid cells, includingicroglia (Barclay et al., 2002). It is suggested that microglia

re maintained in a quiescent state in the intact central ner-ous system by interaction of CD200 with CD200R (Hoekt al., 2000). In support of this, we have recently demon-trated that microglial activation in aged and A�-treatedats was accompanied by decreased expression of CD200n vivo and that A�- and LPS-induced microglial activationn vitro was down-regulated by co-culturing microglia witheurons (as the cellular source of CD200); A� and LPS-nduced IL-1� production in glia decreased 1.7 fold and 3.4old, respectively, in the presence of neurons (Lyons et al.,007a). These data highlight the fact that microglial cellctivity is modulated by CD200 recognition of CD200R.onsistently, we demonstrate that the age-related decrease

n CD200 is accompanied by increased microglial activation

s assessed by CD86 and ICAM-1, and show that FGL atten-ates these changes and the age-related decrease in CD200.his immunomodulatory role for CD200 is consistent withvidence showing that CD200−/− mice exhibit an exag-

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26 E.J. Downer et al. / Neurobi

erated inflammatory response to trauma and an increasedusceptibility to autoimmune disease (Hoek et al., 2000).urthermore, elevated CD200 expression has been shown torotect neurons from microglia-induced damage in vivo andn vitro (Chitnis et al., 2007), while LPS-induced TNF-� pro-uction in myeloid cells was enhanced in CD200R−/− miceBoudakov et al., 2007). While a number of factors otherhan CD200, including neurotrophins and neuropeptides, arenvolved in regulating the expression of markers of activated

icroglia (Aloisi, 2001), the data herein support recent evi-ence from our laboratory demonstrating a central role forD200 in controlling microglial activation status (Lyons etl., 2007a).

The primary source of IL-1� is activated microglia (Lit al., 2003) and our findings show that the age-relatedicroglial activation is accompanied by increased IL-1�

xpression, while FGL attenuates these age-related changesn parallel. This was confirmed by the observation that thePS-induced increase in IL-1� protein was attenuated byGL in both mixed glial cultures and isolated microglial cul-

ures. IL-1� protein is also a secretory product of astrocytesFu et al., 2007), but the data demonstrate that FGL attenu-ted the LPS-induced increase in IL-1� in astrocytes only athe highest concentration. Since FGL binds to both FGFR1Kiselyov et al., 2003) and FGFR2 (Christensen et al., 2006),t has the proclivity to target all cell types expressing FGFR,ncluding microglia (Liu et al., 1998) and astrocytes (Clarket al., 2001), which is shown here. This direct effect of FGLn IL-1� production by these cells indicates a second possi-le mechanism of action, in addition to its ability to promoteL-4-induced neuronal expression of CD200.

The anti-inflammatory role for FGL described heres supported by evidence indicating that FGL attenu-tes microgliosis and astrocytosis induced by A�25–35 asvidenced by CD11b and glial fibrillary acidic proteinGFAP) immunoreactivity in the hippocampus and cortexKlementiev et al., 2007) although FGL does not affect basalxpression of inflammatory markers (CD86, ICAM-1, IL-1�)n hippocampus of young animals. Interestingly, the syntheticCAM peptide C3d inhibits the pro-apoptotic actions of IL-� on pancreatic � cells (Petersen et al., 2006), suggestinghat, in addition to modulating IL-1� release from microglia,CAM mimetic peptides may interfere with IL-1�-inducedro-apoptotic signaling.

We have previously observed an inverse relationshipetween hippocampal concentrations of IL-1� and IL-4nd have proposed that the balance between pro- and anti-nflammatory cytokines determines whether LTP is sustainedVereker et al., 2001; Maher et al., 2005). The present data areonsistent with this, and demonstrate that FGL corrects thembalance between these cytokines and restores LTP in agedats. Therefore, in addition to the age- and FGL-associated

hanges in IL-1�, the age-related decrease in IL-4 protein islso reversed by FGL. In vitro analysis indicating that FGLose-dependently increases IL-4 production in mixed glia inhe presence of LPS is consistent with previous evidence indi-

bi(c

f Aging 31 (2010) 118–128

ating that the cell source of IL-4 in brain is glia (Nolan etl., 2005; Park et al., 2005).

IL-4 has been shown to decrease microglial activationnduced by LPS in vitro and to attenuate the age-relatedncrease in microglial activation in vivo (Lynch et al., 2007;yons et al., 2007b); we have proposed that this is medi-ted by an IL-4-induced increase in CD200 (Lyons et al.,007a). Indeed, IL-4 deficient mice display reduced neuronalD200 expression while exogenous IL-4 enhances CD200

n vitro and in vivo in parallel (Lyons et al., 2007a). Theolecular mechanism controlling CD200 is unknown but ourndings indicate that the IL-4 induction of neuronal CD200,hich is triggered by FGL, is dependent on ERK signaling.

nterestingly, ERK phosphorylation is increased in responseo FGL (Neiiendam et al., 2004) and the MEK inhibitor,D98059, has been shown to attenuate FGL-induced neuriteutgrowth in neurons (Neiiendam et al., 2004) and decrease3d-mediated neuroprotection against the parkinsonian toxin-hydroxydopamine (6-OHDA) (Ditlevsen et al., 2007), indi-ating the biological importance of NCAM-mediated ERKctivation. In support of this, we report that FGL enhancedRK phosphorylation in the hippocampus, reversing the age-

elated decline in ERK expression and the deficit in LTP.uch evidence indicates that ERK plays a role in the expres-

ion of LTP (Giovannini, 2006), and it has been shown that theecrease in LTP in aged rats is accompanied by a decrease inhospho-ERK expression (Maher et al., 2005). Furthermore,e have previously shown that a single intracerebroventricu-

ar injection of IL-4 abrogates the age-related impairment inTP (Nolan et al., 2005), strengthening the suggestion thathe age-related dampening in IL-4/ERK signaling events isivotal in attenuating LTP in the aged rat. Consistent with itsodulatory effect on LTP, FGL induces a long-lasting facil-

tation of performance in two hippocampus-dependent tasksCambon et al., 2004), indicating that the hippocampus is aarget for the FGL peptide.

The data suggest that FGL may be important therapeuti-ally in age-associated neurodegenerative diseases. As far ase are aware this is the first evidence of a role for a drug in reg-lating CD200 expression, and highlights the FGL peptide asn attractive clinical possibility. Our findings do not excludehe possibility that FGL acts on infiltrating leukocytes givenhat FGFR1 expression is localised on T cells (Byrd et al.,996). However, FGL readily crosses the blood–brain barriernd is rapidly detected in the cerebrospinal fluid followingubcutaneous administration (Secher et al., 2006). It is alsooteworthy that data from behavioral tests performed at var-ous time points following FGL administration indicates thatGL induces a prolonged effect, and does not necessarilyeed to be present at the time of test performance (Sechert al., 2006). The findings presented here indicate that FGLan act directly on isolated glia to decrease IL-1� production

ut can also modulate microglial activation by permitting annteraction between neurons and glia. We propose a modelFig. 6) whereby FGL modulates IL-4 production by glialells, which acts on neurons to maintain CD200 expression

E.J. Downer et al. / Neurobiology o

Fig. 6. Model of FGL-induced anti-inflammatory signaling in responseto age and LPS. This model is a schematic representation showing FGLtargeting glial cells to promote anti-inflammatory IL-4 production. IL-4can impact on neurons to regulate CD200 expression via ERK signalingemaC

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vents. By regulating CD200 expression at the neuronal membrane, FGLaintains the CD200–CD200R interaction between neurons and microglia,

nd subsequently attenates microglial activation (IL-1� production andD86/ICAM-1 expression) associated with aging in the hippocampus.

aintaining the interaction between CD200 and CD200R andhereby the microglia in a quiescent state.

cknowledgements

This work was supported by the EU-supported integratedroject PROMEMORIA.

Disclosure: Authors have no conflict of interest to disclose,nancial, personal or otherwise with people or organizationsithin three years of beginning the work submitted that could

nappropriately influence this study.This work has not been published elsewhere and is not

nder review with another journal. The authors have agreedo the submission of the final manuscript.

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