Multiple sclerosis (MS) is a chronic demyelinating diseaseof the CNS leading to permanent cognitive and motordisabilities and characterized by inflammation, demyelina-tion, oligodendrocyte loss, and axonal pathology (Kutzel-nigg et al. 2005; Tumani et al. 2009; Bramow et al.2010). The etiology of MS is unknown and no effectivecure is available. Activation of arachidonic acid metabolicpathway has been reported in MS (Dore-Duffy et al. 1986;Neu et al. 2002), however it is unclear whether it is aconsequence of increased neuroinflammation or if plays arole in the initiation or the progression of demyelination.Arachidonic acid is a n-6 polyunsaturated fatty acid, whichis released upon inflammatory stimuli and then convertedby cyclooxygenase (COX)-1 and -2 to prostaglandins(PGs), potent mediators of inflammation (Farooqui et al.2007). The levels of COX-derived PGI2, PGF2a, PGD2 andparticularly, PGE2, have been found increased in the CSFof MS patients (Dore-Duffy et al. 1986, 1991). COX-2protein expression is also increased in regions of activedemyelination, and co-expressed mostly with markers of

macrophages/microglia (Rose et al. 2004), oligodendro-cytes (Carlson et al. 2006), and apoptosis (Carlson et al.2006). Increased COX-2 expression in demyelinating

Received April 7, 2011; revised manuscript received/accepted May 12,2011.Address correspondence and reprint requests to Dr. Francesca Bosetti,

Brain Physiology and Metabolism Section, National Institute on Aging,National Institutes of Health, 9 Memorial Drive, Room 1S126 MSC0947, Bethesda, MD 20892, USA. E-mail: frances@mail.nih.gov1The present address of Christopher D. Toscano is the Food and DrugAdministration, Center for Drug Evaluation and Research, Office of NewDrugs, Silver Spring, MD 20993, USA.2The present address of Francesca Bosetti is the National Institute ofNeurological Disorders and Stroke, National Institutes of Health, 6001Executive Blvd. Rm. 2118, Bethesda, MD 20892-9521, USA.Abbreviations used: COX, cyclooxygenase; CPZ, cuprizone; DP1,

prostaglandin D2 receptor 1; EAE, experimental autoimmune encepha-lomyelitis; EP, prostaglandin E2 receptor; GFAP, glial fibrillary acidicprotein; MS, multiple sclerosis; NG2, chondroitin sulfate proteoglycan 4precursor; NOGOA, neurite outgrowth inhibitor A or reticulon 4 orRTN4; PDGFR, platelet-derived growth factor alpha receptor; PGs,prostaglandins; TNF-a, tumor necrosis factor alpha.

,1

,2

*Molecular Neuroscience Unit, Brain Physiology and Metabolism Section, National Institute on Aging,

Bethesda, Maryland, USA

�National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda,

Maryland, USA

Abstract

Cyclooxygenases (COX)-1 and -2 are key enzymes required

for the conversion of arachidonic acid to eicosanoids, potent

mediators of inflammation. In patients with multiple sclerosis,

COX-2 derived prostaglandins (PGs) are elevated in the CSF

and COX-2 is up-regulated in demyelinating plaques. How-

ever, it is not known whether COX-2 activity contributes to

oligodendrocyte death. In cuprizone-induced demyelination,

oligodendrocyte apoptosis and a concomitant increase in the

gene expression of COX-2 and PGE2-EP2 receptor precede

histological demyelination. COX-2 and EP2 receptor were

expressed by oligodendrocytes, suggesting a causative role

for the COX-2/EP2 pathway in the initiation of oligodendrocyte

death and demyelination. COX-2 gene deletion, chronic

treatment with the COX-2 selective inhibitor celecoxib, or with

the EP2 receptor antagonist AH6809 reduced cuprizone-

induced oligodendrocyte apoptosis, the degree of demyelin-

ation and motor dysfunction. These data indicate that the

PGE2 EP2 receptor contributes to oligodendrocyte apoptosis

and open possible new therapeutic approaches for multiple

sclerosis.

Keywords: arachidonic acid, cuprizone, cyclooxygenases,

demyelination, EP2, myelin.

J. Neurochem. (2012) 121, 418–427.

JOURNAL OF NEUROCHEMISTRY | 2012 | 121 | 418–427 doi: 10.1111/j.1471-4159.2011.07363.x

418 J. Neurochem. (2012) 121, 418–427Published 2011. This article is a US Government work and is in the public domain in the USA

lesions and increased PGs levels in the CSF of MSpatients suggest that this pathway is involved in thedisease, and studies in various animal models of demye-lination support this concept. In the mouse model ofTheiler’s encephalomyelitis virus-induced demyelination,COX-2 is expressed by mature oligodendrocytes undergo-ing apoptosis (Carlson et al. 2006), suggesting that COX-2plays a role in mediating oligodendrocytes apoptosis. Inthe experimental autoimmune encephalomyelitis (EAE)model, both mixed COX inhibitors, such as indomethacinor naproxen, and COX-2 selective inhibitors inhibitimmune system activation, inflammation and demyelinationin the spinal cord (Reder et al. 1995; Muthian et al. 2006;Ni et al. 2007). Therefore, the efficacy of COX non-selective and COX-2 selective inhibitors could be linked tothe inhibition of the synthesis of PGs, and particularly ofPGE2, which is increased in EAE (Kihara et al. 2009).PGE2 acts via G protein-coupled PGE2 receptors (EP 1, 2,3 and 4) that mediate different signal transductionpathways (Bos et al. 2004). Evidence in the EAE modelsuggests that the PGE2 EP4 receptor-mediated pathway isinvolved in the regulation of T cells differentiation (Yaoet al. 2009). However, the direct involvement ofCOX-2/PGE2/EP pathway in myelin and oligodendrocyteinjury independently of the immune system is not known.

Cuprizone-induced primary demyelination has beenproposed as a valuable model to reproduce patterns IIIand IV of MS active demyelinating lesions, characterizedby oligodendrocyte dystrophy and apoptotic death (Kippet al. 2009; Liu et al. 2010). Oligodendrocytes undergocaspase 3 activation-mediated apoptosis after 1 week ofcuprizone exposure, followed by severe brain demyelina-tion between week 3 and 6, with a peak at week 5 ofcontinuous exposure to a cuprizone-containing diet(Torkildsen et al. 2008; Groebe et al. 2009; Norkute et al.2009; Palumbo et al. 2011). Mice treated with cuprizonefor up to 6 weeks and then returned to regular cuprizone-free diet remyelinate spontaneously (Matsushima andMorell 2001).

To investigate the time-dependent role for COX-2/PGE2

EP receptors signaling pathway, we used COX-2)/) miceand C57BL/6 mice treated with celecoxib or with the EP2antagonist AH6809. Mice were killed either after 1 weekfrom cuprizone exposure to evaluate early changesconcomitant with activation of caspase 3 in oligodendro-cytes, or after 5 weeks of cuprizone exposure, at the peakof demyelination to determine the effects on demyelinationand motor performance (Torkildsen et al. 2008; Palumboet al. 2011). Our data indicate that COX-2 gene deletionor inhibition with celecoxib attenuates apoptotic death ofoligodendrocytes, demyelination and motor dysfunctionand that these effects are mediated by the PGE2 EP2receptor pathway.

Materials and methods

Animal proceduresEight-week-old male C57BL/6 mice (Taconic Farms, Germantown,NY, USA) and COX-2)/) and COX-2+/+ mice with identical strainand genetic backgrounds obtained from heterozygous by heterozygousbreedings (Toscano et al. 2007) were fed ad libitum with a powdereddiet (Purina #5002; Research Diets, New Brunswick, NJ, USA)containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone;Sigma-Aldrich, St. Louis, MO, USA) for 5 weeks to induce asubstantial demyelination in the brain (Torkildsen et al. 2008). Controlmice were fed a control standard diet for 5 weeks. Mice wereanesthetized with pentobarbital 50 mg/kg (Nembutal, Ovation Phar-maceuticals Inc., Lake Forest, IL, USA) and decapitated for molecularanalysis, or were intracardially perfused with a 4% paraformaldehydesolution for histology. All procedures were performed under a NIHapproved animal protocol, in accordance with NIH guidelines.

Pharmacological treatmentsCelecoxib (Celebrex�, 400 mg capsules) was purchased from Pfizer(G.D. Searle LLC, New York, NY, USA). Celecoxib was mixedinto the diet at a concentration of 4000 ppm, together with 0.2%cuprizone (Purina #5002; Research Diets) and mice were fed for1 week or 5 weeks (Toscano et al. 2008). Control mice received acelecoxib-free 0.2% cuprizone diet. Celecoxib is a COX-2 selectiveinhibitor with a selectivity ratio > 375 for COX-2 vs. COX-1(Davies et al. 2000).

The EP2 receptor antagonist AH6809 (Cayman Chemical, AnnArbor, MI, USA) was dissolved in a solution of 25% solutol (BASFCorporation, Iselin, NJ, USA) in sterile 0.9% saline (20% dimethylsulfoxide) at a final concentration of 5 lg/lL and administered oncedaily at a dose of 30 mg/kg by intraperitoneal (i.p.) injection for1 week or 5 weeks. AH6809 binding affinity to the EP2 receptor hasbeen characterized in human and mouse. AH6809 is a non-selectiveantagonist with nearly equal affinity for EP (1, 2 and 3) and PGD2

receptor 1 (DP1) (Abramovitz et al. 2000); whereas in the mouse,AH6809 has the highest affinity for the EP2 receptor and also, showsa weak binding to EP1 and DP1 receptors (Kiriyama et al. 1997).

Myelin stainingThirty-lm coronal brain sections were cut on a cryostat (BrightInstrument Company, LTD, Huntingdon, England). Sections werestained with Black-Gold II (Histo-Chem, Jefferson, AR, USA) aspreviously described (Schmued and Slikker 1999). Sections werecleared in Histo-Clear (National Diagnostics, Atlanta, GA, USA)and coverslipped using DPX (Sigma-Aldrich) mounting medium.Section were photographed (Olympus U-CMAD3 camera, OlympusImaging America Inc., Center Valley, PA, USA) at 10· magnifi-cation between Bregma )0.22 mm and )0.58 mm. Images wereopened with Spot Advanced 4.1 software (SPOTTM ImagingSolution, Sterling Heights, MI, USA) which was used to set theblank (background subtraction). Images were imported into Image Jto measure the mean optical density within the middle of the corpuscallosum, at the level of the fimbria, and of the cortex. Myelindensities for each mouse were normalized against optical densityvalues in unchallenged control mice. n = 6 animals (control groups)and 11 animals [cuprizone (CPZ)-treated groups].

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COX-2/PGE2/EP2 pathway during demyelination | 419

Prostaglandin measurementLipid extraction was performed using 3 : 2 hexane/2-propanol aspreviously described (Aid and Bosetti 2007). Prostaglandin levelswere determined by ELISA-based assays (Oxford BiomedicalResearch Kit (Burington, ON, Canada, for PGE2 and TXB2;Cayman Chemical for PGD2). Prostanoids levels were determinedfollowing the manufacturer’s instructions. n = 5 animals (controlgroups) and 6 animals (CPZ-treated groups).

ImmunohistochemistryFloating sections were treated following the Vectastain ABCprotocol (Vector Laboratories, Burlingame, CA, USA). Anti-PDGFRa antibody (platelet-derived growth factor alpha receptor10 lg/mL; Millipore, Temecula, CA, USA) was used as a markerfor oligodendrocyte precursors, as previously reported (Hall et al.1996; Wilson et al. 2006). n = 4 animals (control groups) and 6animals (CPZ-treated groups).

ImmunofluorescenceThirty-lm coronal brain fixed sections were incubated for 1 h in ablocking buffer (phosphate-buffered saline 1% bovine serumalbumin, 0.1% Triton-X 100), and incubated overnight at 4�C withthe following primary antibodies: anti-NOGO-A clone 6A5.2(1 : 750; Millipore), cleaved caspase 3 Asp175 (1 : 400, CellSignaling, Danvers, MA, USA) and EP2 (1 : 200, CaymanChemical). Sections were incubated for 1 h with the followingAlexa Fluor fluorochrome-conjugated secondary antibodies in thedark at 25�C: 594 goat anti-mouse IgG, 488 goat anti-rabbit IgG(5 lg/mL) (Invitrogen, Carlsbad, CA, USA). VECTASHIELD�

Mounting Medium with DAPI (Vector Laboratories) was used.n = 3 animals/group. Stained sections were imaged with a Fluoview1000 confocal microscope (Olympus America Inc., Center Valley,PA, USA) in the corpus callosum. All images were acquired using aUPLSAPO ·10 numerical aperture 0.4 dry objective and aPLANAPO ·60 numerical aperture 1.4 oil immersion objective(Olympus America Inc.). Images were processed using Imaris 5.7(Bitplane Inc., South Windsor, CT, USA) and assembled usingAdobe Photoshop CS (Adobe Systems Incorporated, McLean, VA,USA).

Caspase 3 activity testCorpus callosum was manually dissected from fresh brainsimmediately after decapitation and stored at )80�C. Protein wereextracted with an homogenization buffer containing 50 mM Tris-HCl, 5 mM EDTA, 2 mM EGTA, 2 mM DTT, protease andphosphatase inhibitors. Samples were homogenized and centrifuged20 min at 1 400 g at 4�C. Supernatant was collected and caspase 3activity was measured using a Caspase 3 Colorimetric ActivityAssay Kit (Chemicon, Temecula, CA, USA) as directed by themanufacturer. Results were expressed as total amount of pNA (lM).n = 3 animals (control groups) and 4 animals (CPZ-treated groups).

Quantitative real time RT-PCRRT-PCR was performed using cDNA form the cerebral cortex, withthe ABI PRISM 7000 Sequence Detection System (AppliedBiosystems, Life Technologies Corporation, Carlsbad, CA, USA).Results were normalized to phosphoglycerate kinase 1(Mm_00435617_m1) expression levels, as previously reported

(Toscano et al. 2007). Gene expression was analyzed using thefollowing Assays on Demand: glial fibrillary acidic protein (GFAP;Mm01253033_m1); tumor necrosis factor alpha (TNF-a;Mm00434228_m1), EP1 (Mm00443097_m1) EP2 (Mm00436051_m1), EP4 (Mn00436053_m1). The amount of target gene expressionwas calculated by using the DDCT method. n = 4 animals (controlgroups) and 6 animals (CPZ-treated groups).

Rota-rod testUgo Basile Rota-Rod 47600 (Biological Research Apparatus,Comerio, Italy), was used to test motor coordination and balanceafter 5 weeks of cuprizone. All mice were evaluated on the rota-rodthree times a day, as previously described for three consecutive dayswith the rotation set at 16, 24 and 32 rpm. The number of falls (fallsfrom the cylinder) and flips (when the animal hangs on to thecylinder) were recorded within 60 s at 16, 24 and 32 rpm incelecoxib (per os 4000 ppm or i.p. 30 mg/kg) or AH6809-treatedmice and in COX-2)/) mice and in their respective control mice(Franco-Pons et al. 2007). n = 5 animals (control groups) and 10animals (CPZ-treated groups).

StatisticsData from PCR, ELISA, Black-Gold II staining and immunohis-tochemistry were analyzed with a two-way ANOVA for COX-2+/+and )/) mice and by one-way ANOVA for C57BL/6 mice treatedwith celecoxib or AH6809. Bonferroni’s test was used for multiplecomparisons. Rota-rod data were analyzed with one-way ANOVA

non-parametric Kruskal–Wallis test. Dunns post-test was used formultiple comparisons. Data were expressed as mean ±SEM, p value < 0.05 were considered statistically significant(*p < 0.05, **p < 0.01, ***p < 0.001).

Results

COX-2 gene deletion or chronic treatment with celecoxibreduces cuprizone-induced demyelinationmRNA level of COX-2 was increased (p < 0.05) inthe cortex after 1 week of cuprizone administration, andremained increased (p < 0.05) at week 5 of cuprizoneexposure (Figure S1a). COX-2 protein expressionco-localized with the oligodendrocyte marker NOGOA(neurite outgrowth inhibitor A, reticulon 4, RTN4) at thebeginning of apoptotic death of oligodendrocytes (Fig-ure S1b), suggesting a role for COX-2 in oligodendrocyteapoptosis. Supporting this idea, treatment with the COX-2selective inhibitor celecoxib (30 mg/kg) for 5 weeksduring cuprizone intoxication reduced myelin loss in thecorpus callosum and in the cortex, as shown by Black-Gold II staining of myelin (p < 0.001 and p < 0.05,respectively, Fig. 1a and c). Daily intraperitoneal admin-istration of celecoxib 30 mg/kg was performed to confirmthe effect of the oral administration (Figure S2a and b).Similar results were observed in COX-2)/) mice, whichdeveloped less demyelination than COX-2+/+ mice after5 weeks of cuprizone (p < 0.001, Fig. 1b and d). In the

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420 | S. Palumbo et al.

cortex, only COX-2+/+ mice developed a significantdemyelination (p < 0.001).

We tested weather the use of celecoxib later duringcuprizone treatment could be protective against cuprizone-induced demyelination. When celecoxib was administeredstarting at week 1 of cuprizone exposure, it was protectiveagainst demyelination (Figure S3). In opposite, when celec-oxib was administered later, starting at week 4 of cuprizone,when demyelination is already severe, it was not protectiveindicating that an early intervention is necessary to preventdevelopment of demyelination (Figure S3).

Celecoxib treatment reduces markers ofneuroinflammation after cuprizone exposureGene expression of markers of neuroinflammation, such asthe astrocyte marker GFAP, and the proinflammatorycytokine TNF-a were increased by cuprizone exposure inC57BL/6 mice (p < 0.001) (Fig. 2a and b) and in bothCOX-2)/) and COX-2+/+ mice (p < 0.05, p < 0.001)(Fig. 2c and d); however, GFAP expression was attenuatedin celecoxib-treated mice (p < 0.001) (Fig. 2a) and inCOX-2)/) compared to COX-2+/+ mice (p < 0.01)(Fig. 2c). Cuprizone-induced increase in GFAP geneexpression suggests a primary role for astrocytes incuprizone-induced neuroinflammatory response since no

change was found in the relative mRNA level of microgliamarker CD11b (data not shown). Celecoxib treatment alsosignificantly reduced TNF-a gene expression (p < 0.001)(Fig. 2b).

The inflammatory reaction that accompanies cuprizone-induced demyelination is characterized also by the influxof oligodendrocyte precursors into the demyelinated sites(Liu et al. 2010). We confirmed that the number ofPDGFRa+ cells was significantly increased after 5 weeksof cuprizone exposure (p < 0.01) compared to controls(Fig. 2e–j). Chronic treatment with celecoxib, as well asCOX-2 gene deletion, significantly attenuated the influx ofoligodendrocyte precursors (p < 0.05, p < 0.001) (Fig. 2gand j), suggesting reduced inflammation. In addition,COX-2)/) mice did not experience any significantincrease in PDGFR+ cells in response to cuprizone(Fig. 2h). The experiment was duplicated using thechondroitin sulfate proteoglycan 4 precursor (NG2) markerof oligodendrocytes to confirm these results (Figure S4).

Cyclooxygenase-derived proinflammatory prostanoidssuch as PGE2 (p < 0.001), PGD2 (p < 0.05) and TXB2

(p < 0.05) were increased in the cortex after 5 weeks ofcuprizone exposure (Fig. 3a and c), whereas PGI2 levelswere not significantly changed (Figure S5a). Chronic treat-ment with celecoxib selectively suppressed cuprizone-

Fig. 1 Myelin staining after 5 weeks of cuprizone in celecoxib-treated

and in COX-2)/) mice. Black-Gold II staining of myelin and myelin

optical density (score) in the corpus callosum and cortex after 5 weeks

of cuprizone of celecoxib-treated mice (a,c) and COX-2)/) mice (b,d)

compared to controls. Myelin score is reported as % of controls

(100%). Data are means ± SEM (*p < 0.05, ***p < 0.001, #p < 0.05).

4· images; scale bars = 0.20 mm (a–b), 0.10 mm (c–d). n = 6 animals

(control groups) and 11 animals (CPZ-treated groups). Statistics: one-

way ANOVA for celecoxib treatment and two-way ANOVA for COX-2+/+

and )/) mice followed by Bonferroni’s post-test.

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COX-2/PGE2/EP2 pathway during demyelination | 421

induced increase in PGE2 (p < 0.01), but not in the otherprostanoids examined (Fig. 3a and c). Next, we examinedcortical gene expression of PGE2 EP1–4 receptors after1 week or 5 weeks of cuprizone exposure. We found thatonly the EP2 receptor was increased after 1 week ofcuprizone exposure (p < 0.05), paralleling the increase inCOX-2 expression (Fig. 3d). After 5 weeks of cuprizoneintoxication, EP2 and also EP1 and EP4 receptors geneexpression was up-regulated (p < 0.001) (Fig. 3d and f),whereas EP3 receptor gene expression was not changed ateither time point examined (Figure S5b). Moreover, immu-nohistochemistry showed co-localization of the EP2 receptorwith the oligodendrocyte marker NOGOA after 1 week ofcuprizone (Fig. 3j). Co-localization with the oligodendrocytemarker 2¢, 3¢-cyclic nucleotide 3¢-phosphodiesterase or-phosphohydrolase (CNPase) was also observed (Figure S6a).The cuprizone-induced increase of EP1 and EP2 receptorsexpression was not observed in COX-2)/) mice (Fig. 3g andh), supporting a reduction of inflammation along with reduceddemyelination.

COX-2)/) mice and mice treated with celecoxib showimproved motor coordination and balance after cuprizoneexposureMice fed a 0.2% cuprizone diet showed reduced motorcoordination on the rota-rod after 5 weeks of cuprizoneintoxication, which represents the time point of maximum

demyelination (Franco-Pons et al. 2007). Chronic treatmentwith celecoxib reduced the number of falls and flips at32 rpm on the rota-rod compared to cuprizone-exposedcontrol mice (p < 0.05) (Fig. 4a). Similarly, COX-2)/) micedid not develop significant motor dysfunction after 5 weeksof cuprizone administration compared to COX-2+/+ mice(p < 0.05) (Fig. 4b). The progressive evolution of motordeficit during the training trials is reported in Figure S7.

Reduction of oligodendrocyte apoptosis via caspase 3 inCOX-2)/) miceIn agreement with a previous report (Hesse et al. 2010), wefound that oligodendrocytes (NOGOA marker) expresscleaved caspase 3 after 1 week of cuprizone exposure(Fig. 5a, right panels). Parallel immunohistochemistry showedthat oligodendrocytes from age-matched COX-2)/) micewere not caspase 3 positive, suggesting that oligodendrocyteapoptosis via caspase 3 is inhibited in COX-2)/) mice(Fig. 5a, left panels). Next, we measured caspase 3 activityafter 1 week of cuprizone exposure and found that it wassignificantly reduced in COX-2)/) compared to COX-2+/+mice (p < 0.05) (Fig. 5b). Co-localization of caspase 3with the oligodendrocyte marker 2¢, 3¢-cyclic nucleotide3¢-phosphodiesterase or -phosphohydrolase (CNPase) wasalso observed in COX-2+/+ mice (Figure S6b). A decrease inapoptosis in the corpus callosum of COX-2)/) mice wasconfirmed with Tunel stain (Figure S6c).

(a)

(b) (d)

(c) (e)

(f)

(g)

(h)

(i)

(j)

Fig. 2 Markers of neuroinflammation after 5 weeks of cuprizone in

celecoxib-treated and COX-2)/) mice. Changes of mRNA levels of

GFAP and TNF-a induced by cuprizone in celecoxib-treated (a–b) and

COX-2)/) mice (c–d) compared to controls. Number of PDGFRa

positive cells in celecoxib-treated (e) and COX-2)/) mice (h) com-

pared to controls, counted in the corpus callosum. PDGFRa immu-

noreactivity in the corpus callosum of celecoxib-treated mice (g) and

COX-2)/) mice (j) after 5 weeks of cuprizone compared to their

respective cuprizone-treatment control (f and i, respectively). Data are

means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05). 40·images; scale bar = 0.10 mm. n = 4 animals (control groups) and 6

animals (CPZ-treated groups). Statistics: one-way ANOVA for celecoxib

treatment and two-way ANOVA for COX-2+/+ and )/) mice, followed by

Bonferroni’s post-test.

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422 | S. Palumbo et al.

The EP2 receptor antagonist AH6809 reducesdemyelination, motor dysfunction and caspase 3 activityafter cuprizone exposureSimilarly to COX-2 expression, the EP2 receptor geneexpression was increased after 1 week of cuprizone exposure(Fig. 3e). EP2 immunoreactivity was found in oligodendro-cytes concomitant to the detection of the first signs of

apoptosis, suggesting that the downstream effects of COX-2activity on cuprizone-induced oligodendrocyte apoptosiswere EP2-mediated (Fig. 3j). Treatment with the EP2receptor antagonist AH6809 (30 mg/kg, i.p. once daily)during the first week of cuprizone exposure reduced totalcaspase 3 activity (p < 0.01) (Fig. 6d), indicating reducedapoptosis. Furthermore, treatment with AH6809 during theentire 5 weeks of cuprizone intoxication increased myelinscore in the corpus callosum (p < 0.05) and in the cortex(p < 0.05) compared to cuprizone-exposed untreated mice,as shown by Black-Gold II staining (Fig. 6a and b).Treatment with AH6809 for 5 weeks also reduced thenumber of falls and flips on the rota-rod in cuprizone-exposed mice (p < 0.05) (Fig. 6c).

Discussion

We demonstrated that chronic treatment with the COX-2inhibitor celecoxib or with the EP2 receptor antagonistAH6809 reduced cuprizone-induced oligodendrocyte apo-ptosis, demyelination, neuroinflammation, and motor deficits,and indicated that the PGE2 EP2 receptor is the effector of thetoxic effects of COX-2 activity during cuprizone exposure.

Fig. 3 Expression of prostanoids and PGE2 EP receptors. Cortical

levels of PGE2, PGD2 and PXB2 after 5 weeks of cuprizone in

celecoxib-treated mice (a–c). n = 5 animals (control groups) and 6

animals (CPZ-treated groups). Relative cortical gene expression of

EP1, 2 and 4 receptors after 1 and 5 weeks of cuprizone (d–f).

Cortical gene expression of EP1, 2 and 4 in COX-2)/) mice after

5 weeks of cuprizone compared to controls (g–i). n = 4 animals

(control groups) and 6 animals (CPZ-treated groups). EP2 (green)

co-localization with NOGOA (red) after 1 week of cuprizone com-

pared to control (j). 60· confocal images; scale bar = 0.50 lm. Data

are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05).

Statistics: one-way ANOVA for celecoxib treatment and two-way

ANOVA for COX-2+/+ and )/) mice, followed by Bonferroni’s

post-test.

Fig. 4 Motor performance on the rota-rod after 5 weeks of cuprizone.

Number of falls and flips is significantly decreased after 5 weeks of

cuprizone in celecoxib-treated mice (a) and in COX-2)/) mice (b)

compared to controls. n = 5 animals (control groups) and 10 animals

(CPZ-treated groups). Data are means ± SEM (*p < 0.05, **p < 0.01,#p < 0.05). Statistics: one-way ANOVA non-parametric Kruskal–Wallis

test followed by Dunns post-test.

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COX-2/PGE2/EP2 pathway during demyelination | 423

We found that gene expression of both COX-2 and theEP2 receptor, but not other EP receptors, in the cerebralcortex was increased early, after 1 week of cuprizoneintoxication, prior to histological detection of demyelination.COX-2 and EP2 protein expression also became detectable inoligodendrocytes after 1 week of cuprizone exposure, whenoligodendrocytes begin undergoing caspase 3-mediatedapoptosis, suggesting a possible causative role for theCOX-2/EP2 pathway in oligodendrocyte death. Caspase 3activation is a critical early event leading to cuprizone-induced oligodendrocyte apoptosis, and occurs in parallel tothe induction of COX-2 in oligodendrocytes, suggesting thatCOX-2 activity is involved in triggering caspase 3 activation.Evidence from in vitro studies supports a link between COX-2 expression and oligodendrocyte apoptosis and demyelin-ation (Carlson et al. 2010). In COX-2)/) mice, total caspase3 activity in the corpus callosum was inhibited and thespecific activation of cleaved caspase 3 in oligodendrocyteswas suppressed.

Both COX-2)/) mice and C57BL/6 mice treated withcelecoxib or with AH6809 were protected from myelin loss

in the cortex and corpus callosum after 5 weeks ofcuprizone, compared to their respective controls, indicatingan active role for the COX-2 EP2 pathway in the progres-sion of demyelination. These effects were COX-2 selective,as COX-1)/) mice did not show any significant differencein myelin score compared to their respective wild-typecontrols after cuprizone exposure, indicating that COX-1does not play a role in the pathogenesis or exacerbation ofdemyelination in this model (Figure S8). In addition, geneexpression of the astrocyte marker GFAP was reduced inboth COX-2)/) and celecoxib-treated mice; additionally,

Fig. 5 Activation of the proapoptotic protein caspase 3 in oligoden-

drocytes after 1 week of cuprizone. Double immunofluorescence of

oligodendrocytes (NOGOA, red) and activated caspase 3 (green) in

the corpus callosum of COX-2+/+ and COX-2)/) cuprizone-treated

mice (a). (60· confocal images; scale bar = 0.20 lm). Caspase 3

activity expressed by pNA concentration, in COX-2+/+ and COX-2)/)mice (b). n = 3 animals (control groups) and 4 animals (CPZ-treated

groups). Data are means ± SEM (**p < 0.01, #p < 0.05). Statistics:

two-way ANOVA followed by Bonferroni’s post-test.

Fig. 6 Effect of AH6809 treatment on demyelination, motor perfor-

mance and apoptosis. Black-Gold II staining of myelin and myelin

score in the corpus callosum (a) and in the cortex (b) after 5 weeks of

cuprizone in AH6809-treated mice. Myelin score is reported as % of

controls (100%). 4· images; scale bars = 0.20 mm (a), 0.10 mm (b).

Rota-rod test after 5 weeks of cuprizone in AH6809-treated mice

shows number of falls and flips (c). Caspase 3 activity, expressed by

pNA concentration, after 1 week of cuprizone in AH6809-treated mice

(d). Data are means ± SEM (*p < 0.05, ***p < 0.001, #p < 0.05). n = 4

animals/group. Statistics: one-way ANOVA followed by Bonferroni’s

post-test.

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celecoxib treatment also reduced cuprizone-induced TNF-aexpression.

PGE2 emerged as the most affected prostaglandin in MSpatients and in animal models of demyelination (Greco et al.1999; Kihara et al. 2009). In EAE, PGE2 levels are elevatedand correlate with the degree of disability whereas PGI2 andPGD2 levels were decreased (Kihara et al. 2009). We foundthat cuprizone increased PGE2, PGD2 and TXB2 levels in thecortex, with the increase in PGE2 being the more robust.Moreover, selective COX-2 inhibition with celecoxib com-pletely suppressed cuprizone-induced increase in PGE2

levels, suggesting that in this model PGE2 is the mainprostaglandin mediating the COX-2 effect.

Among the four specific PGE2 EP1–4 receptors, only EP2expression gene expression was up-regulated after 1 week ofcuprizone when it was also induced in oligodendrocytes. EP1and EP4 receptors gene expression was up-regulated after5 weeks of cuprizone concomitant with severe demyelina-tion, astrogliosis and microglia activation (Figures S9 andS10) (Matsushima and Morell 2001), suggesting that thesereceptors contribute to the inflammatory response induced bycuprizone. Other reports showed that EP1 and EP4 can beexpressed by microglia and EP4 is expressed also byastrocytes (Carlson et al. 2009; Shi et al. 2010). EP2receptor is also expressed by microglia and astrocytes,which is consistent with the further increase in its geneexpression after 5 weeks of cuprizone exposure compared to1-week exposure (Shi et al. 2010).

Cyclooxygenase-2/PGE2 signaling has been implicated inmechanisms of many brain diseases, including Alzheimer’sdisease and ischemia. Specifically, activation of EP2 receptorregulated mechanisms of microglia internalization and micro-glia-induced neurotoxicity (Cimino et al. 2008). In contrast,PGE2 signaling via EP2 and EP4 was neuroprotective duringexcitotoxicity in neurons (Bilak et al. 2004). The neuropro-tective versus neurotoxic effects of EP2 receptor signalingmay depend on the specific cell type affected and on the typeof injury. In our study, EP2 receptor was expressed byoligodendrocytes in an early stage of cuprizone intoxicationsuggesting that EP2 receptor is activated concomitantly tooligodendrocytes apoptosis. However, EP2 could also con-tribute to oligodendrocytes injury by modulating secretion ofproinflammatory cytokines by microglia (Pang et al. 2010).Since AH6809 also shows a weak binding to EP1 and DP1receptors, we cannot exclude that a partial inhibition of EP1and DP1 receptors might contribute to AH6809 beneficialeffect against cuprizone-induced demyelination.

It is important to investigate early events in the demyelin-ation process in order to understand the mechanisms under-lying the pathogenesis of demyelinating diseases. Humanstudies indicate that apoptosis of oligodendrocytes is an earlyevent preceding the formation of plaques in MS (Barnett andPrineas 2004). Our study suggests that selective COX-2inhibitors may be useful in the early treatment of MS to reduce

the degree of demyelination and motor dysfunction. Further-more, considering that the use of selective COX-2 inhibitorshas been associated with adverse cardiovascular events (Hinzet al. 2007), our study suggests that EP2 receptor antagonismcould be an alternative, effective and more specific therapeuticstrategy to attenuate demyelination and improve motorperformance. Currently there are no clinical trials usingCOX-2 selective inhibitors in MS patients. One clinical trialin MS showed that aspirin, a mixed COX inhibitor,improved the severity of MS-related fatigue (Wingerchuket al. 2005). Symptoms of MS such as fatigue and motordisabilities have to be considered because they drasticallyinfluence the quality of life of MS patients.

In conclusion, in cuprizone-induced demyelination COXisoforms have different impact on the demyelination process,with COX-2 being selectively involved in the initiation andprogression of demyelination via the modulation of caspase 3mediated apoptosis by a PGE2 EP2 receptor signalingmechanism. Thus, selective COX-2 inhibitors or EP2 receptorantagonists may be useful as an early treatment to reduce theextent of demyelination and the severity of motor disabilities.

Acknowledgements

This research was supported by the Intramural Research Program ofthe NIH, National Institute on Aging and National Institute ofDental and Craniofacial Research.

Supporting information

Additional supporting information may be found in the onlineversion of this article:

Figure S1. COX-2 gene and protein expression induced bycuprizone.

Figure S2. Intra peritoneal administration of celecoxib exertsprotective effect against cuprizone-induced demyelination andmotor deficit.

Figure S3. Celecoxib treatment modifies early mechanisms ofdemyelination.

Figure S4. Number of NG2 positive cells in the corpus callosumof celecoxib-treated (a) and COX-2)/) mice (d) compared tocontrols, NG2 immunoreactivity in the corpus callosum of celec-oxib-treated mice (b–c) and of COX-2)/) mice (e–f) compared tocontrols.

Figure S5. PGI2 levels in the cerebral cortex of celecoxib-treatedmice after 5 weeks of cuprizone (a). Relative gene expression ofEP3 receptor in the cerebral cortex after 1 and 5 weeks of cuprizone (b).

Figure S6. Double immunofluorescence of oligodendrocytes(CNPase, red) and EP2 receptor (green) in the corpus callosum after1 week of cuprizone (a). Double immunofluorescence of oligoden-drocytes (CNPase, red) and activated caspase 3 (green) in the corpuscallosum after 1 week of cuprizone (b). (c) Tunel stain in the corpuscallosum after 1 week of cuprizone of COX-2)/) mice compared toCOX-2+/+ mice.

Figure S7. Time dependent changes of the motor performance atthe rota-rod after 5 weeks of cuprizone. Number of falls and flips at

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16, 24 and 32 rpm, was recorded for three consecutive days, duringthe last three days of cuprizone administration in celecoxib-treated(a-c) and COX-2)/) mice (d-f) compared to controls.

Figure S8. Black-Gold II staining of myelin the corpus callosum(a) and in the cortex (b) of COX-1)/) mice after 5 weeks ofcuprizone.

Figure S9. Progressive demyelination and gliosis in the corpuscallosum induced by cuprizone.

Figure S10. Astrogliosis and microglia activation after 5 weeksof cuprizone.

As a service to our authors and readers, this journal providessupporting information supplied by the authors. Such materials arepeer-reviewed and may be re-organized for online delivery, but arenot copy-edited or typeset. Technical support issues arising fromsupporting information (other than missing files) should beaddressed to the authors.

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