Prostacyclin promotes oligodendrocyte precursor recruitment and remyelination after spinal cord demyelination

Abstract

Adult oligodendrocyte precursor cells (OPCs) are located adjacent to demyelinated lesion and contribute to myelin repair. The crucial step in remyelination is the migration of OPCs to the demyelinated area; however, the mechanism of OPC migration remains to be fully elucidated. Here we show that prostacyclin (prostaglandin I ₂ , PGI₂ ) promotes OPC migration, thereby promoting remyelination and functional recovery in mice after demyelination induced by injecting lysophosphatidylcholine (LPC) into the spinal cord. Prostacyclin analogs enhanced OPC migration via a protein kinase A (PKA)-dependent mechanism, and prostacyclin synthase expression was increased in the spinal cord after LPC injection. Notably, pharmacological inhibition of prostacyclin receptor (IP receptor) impaired remyelination and motor recovery, whereas the administration of a prostacyclin analog promoted remyelination and motor recovery after LPC injection. Our results suggest that prostacyclin could be a key molecule for facilitating the migration of OPCs that are essential for repairing demyelinated areas, and it may be useful in treating disorders characterized by demyelination.

OPEN

Citation: Cell Death and Disease (2013) 4, e795; doi:10.1038/cddis.2013.335

& 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13 http://www.nature.com/cddis

Web End =www.nature.com/cddis

C Takahashi1,2, R Muramatsu*,1,2, H Fujimura3, H Mochizuki2,4 and T Yamashita*,1,2

Adult oligodendrocyte precursor cells (OPCs) are located adjacent to demyelinated lesion and contribute to myelin repair. The crucial step in remyelination is the migration of OPCs to the demyelinated area; however, the mechanism of OPC migration remains to be fully elucidated. Here we show that prostacyclin (prostaglandin I2, PGI2) promotes OPC migration, thereby promoting remyelination and functional recovery in mice after demyelination induced by injecting lysophosphatidylcholine (LPC) into the spinal cord. Prostacyclin analogs enhanced OPC migration via a protein kinase A (PKA)-dependent mechanism, and prostacyclin synthase expression was increased in the spinal cord after LPC injection. Notably, pharmacological inhibition of prostacyclin receptor (IP receptor) impaired remyelination and motor recovery, whereas the administration of a prostacyclin analog promoted remyelination and motor recovery after LPC injection. Our results suggest that prostacyclin could be a key molecule for facilitating the migration of OPCs that are essential for repairing demyelinated areas, and it may be useful in treating disorders characterized by demyelination.

Cell Death and Disease (2013) 4, e795; doi:http://dx.doi.org/10.1038/cddis.2013.335

Web End =10.1038/cddis.2013.335 ; published online 12 September 2013

Subject Category: Neuroscience

Oligodendrocytes form myelin sheaths around axons that are essential for the high-speed transmission of electrical impulses1 and for providing trophic support to axons.2Damage to oligodendrocyte lead to impairments in the motor, sensory, cognitive, and other neuronal functions.3 Multiple sclerosis (MS) is associated with multifocal demyelination and oligodendrocyte injury in the central nervous system (CNS).The proposed mechanisms of demyelination in MS vary depending on disease stage. During early stages, new myelin forms spontaneously in response to myelin degradation.4,5

The new myelin forms a thin sheath with short internodes that contribute to restoring rapid saltatory conductance in the CNS and preventing axonal damage.6 In the late chronic stages, remyelination failure is a prominent pathologic feature that probably contributes to the relentless progression of the disease.7 Therefore, determining how remyelination occurs in white matter under pathologic conditions may identify new therapeutic avenues to explore to treat CNS disease.

Myelination is mediated by oligodendrocyte precursor cells (OPCs) that are widely distributed throughout both the gray and white matter of the CNS throughout life.8,9 Adult-born oligodendrocyte can continue to proliferate and produce

Keywords: lysophosphatidylcholine; protein kinase A; multiple sclerosis

Abbreviations: OPC, oligodendrocyte progenitor cell; PGI2, prostacyclin I2; MS, multiple sclerosis; CNS, central nervous system; MBP, myelin basic protein; DMEM,

Dulbeccos Modied Eagle medium; PLL, poly-L-lysine; PDGF, platelet-derived growth factor; PFA, paraformaldehyde; PBS, phosphate-buffered saline; PKA, protein kinase A; PGIS, prostacyclin synthase; Th, thoracic; DAPI, 40,6-diamidino-2-phenylindole; BMS, basso mouse scale; SEM, standard error of the mean; ANOVA, analyses of variance; cAMP, cyclic adenosine monophosphate; IL, interleukin; IFN, interferon; EAE, experimental autoimmune encephalomyelitis; cPLA2a, cytosolic phospholipase A2a; mPGES-1, microsomal prostaglandin E synthase-1

Received 15.7.13; accepted 07.8.13; Edited by A Verkhratsky

Prostacyclin promotes oligodendrocyte precursor recruitment and remyelination after spinal cord demyelination

compact myelin.9 In response to demyelinating insults,

A2B5-positive (A2B5) OPCs subsequently proliferate, migrate, and attain maturity to become O4 premyelinating oligodendrocyte before differentiating into mature myelin basic protein (MBP)-positive myelinating oligodendrocytes in the demyelinated lesion. OPCs that are recruited to the lesioned area differentiate into mature remyelinating oligodendrocyte and are engaged in the formation of new myelin sheaths around axons.10 However, demyelinated MS plaques vary with regard to oligodendrocyte content,11 and

some are characterized by a lack of OPCs, which is considered to be due to impaired recruitment of OPCs to demyelinated areas.7 To answer this signicant question, it is important to elucidate the mechanism of OPC recruitment in the adult CNS.

Prostacyclin (prostaglandin I2, PGI2) is a predominant cyclooxygenase metabolite of arachidonic acid synthesized by vascular endothelial and smooth muscle cells. Prostacyclin acts as primary vasodilator as well as an inhibitor of leukocyte adhesion and platelet aggregation.12,13 Recent studies have

emphasized that prostacyclin is not solely a regulator of the cardiovascular system; it is also involved in a diverse set of functions under homeostatic and pathological conditions in

1Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 5650871, Japan; 2Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Chiyoda, Tokyo 1020075, Japan; 3Toneyama National Hospital, Toyonaka, Osaka, Japan and 4Department of Neurology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan*Corresponding authors: R Muramatsu or T Yamashita, Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected] or mailto:[email protected]

Web End [email protected]

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Figure 1 Prostacyclin analogs promote OPC migration in vitro. (a) Expression of IP receptor in A2B5 OPC obtained from a postnatal day 1 mouse. Scale bar, 100 mm. (b) Western blot analyses for IP receptor (upper panel) and a-tubulin (lower panel) in cultured OPCs (left) and cortical neurons (right). (c and d) Cell migration assay showing the dose response to prostacyclin analogs in OPCs incubated with the indicated concentration of iloprost (prostacyclin analog) (c) or cicaprost (prostacyclin analog)

(d). The graphs show the relative number of cells that migrated to the lower chamber after 16 h of culture. (e and f) The number of OPCs that were cultured under indicated conditions as described in Figures 1c and d. Values represent the meanS.E.M. (n 3). *Po0.05 and **Po0.01 compared with control with individual prostacyclin analogs

the CNS. Both prostacyclin synthase (PGIS) and prostacyclin receptor are widely distributed in the CNS,14,15 and prostacy

clin signaling has been demonstrated to protect neurons against ischemic damage.16,17 We previously showed that prostacyclin derived from neovessels accelerates axonal rewiring, thereby enhancing motor recovery following CNS injury.18 These ndings prompted us to test the possibility that prostacyclin contributes to CNS remyelination caused by demyelination disorders.

Here, we studied the role of prostacyclin in OPC recruitment into demyelinated lesions. An in vitro migration assay revealed that prostacyclin promoted OPC migration without inducing cell proliferation. In the lysophosphatidylcholine (LPC)-induced focal spinal cord demyelination mouse model, protein expression of PGIS was increased. Treatment with IP receptor antagonist prevented both spontaneous OPC recruitment into the lesion site and functional recovery following LPC injection. Furthermore, treatment with a prostacyclin analog promoted OPC recruitment and improved

demyelination-induced motor disability. Thus, prostacyclin could be a player in OPC recruitment and may serve as a promising molecule to treat demyelination diseases.

Results

Prostacyclin promotes OPC migration in vitro. We rst examined the expression of prostacyclin receptor (IP receptor) in OPCs. We puried the A2B5 OPCs from postnatal day 1 mice. Immunocytochemical analysis demonstrated that IP receptor was expressed in A2B5 OPCs, which were cultured for 2 days in vitro (Figure 1a). IP receptor expression was also conrmed by western blotting of lysates from both OPC and cortical neuron (positive control) (Figure 1b). We then assessed whether IP receptor expression is associated with OPC migration. OPCs were plated onto the transwell culture insert containing stable prostacyclin analog, iloprost or cicaprost. After 16 h, we counted the number of OPCs that migrated across the transwell membranes and determined

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Figure 2 cAMP and PKA signal are required for prostacyclin-induced OPC migration. (a and b) Quantication of the OPC migration under each treatment. Cells were preincubated with Rp-cAMPS, which is a PKA antagonist (100 mM) and then cultured with iloprost (1 mM, prostacyclin analog) or cicaprost (1 mM, prostacyclin analog).

Sp-cAMPS is a PKA agonist (100 mM). (c and d) The graph shows the relative number of OPCs that were treated with indicated agents as described in Figures 2a and b. Values represent the meanS.E.M. (n 3). **Po0.01 compared with control, ##Po0.01 compared with iloprost or cicaprost treatment

that the prostacyclin analogs promoted OPC migration (Figures 1c and d). To exclude the possibility that the increased number was due to increased cell proliferation, we counted the total number of OPCs in the cultures. There was no signicant difference in cell number under any of the treatments (Figures 1e and f). These results demonstrated that iloprost and cicaprost enhanced OPC migration in culture.

PKA is required for prostacyclin-induced OPC migration. IP receptor is a G protein-coupled receptor that stimulates adenylyl cyclase to synthesize adenosine 30 50-monophosphate (cyclic adenosine monophosphate, cAMP) from adenosine triphosphate.19 Because cAMP elevation and protein kinase A (PKA) activation have been shown to be required for efcient cell migration,20 we predicted that prostacyclin-mediated OPC migration would be dependent on PKA activation. Treatment with the PKA antagonist Rp-cAMPS abolished prostacyclin analog-mediated OPC migration (Figures 2a and b). Conversely, treatment with Sp-cAMPS, a PKA agonist, enhanced OPC migration (Figures 2a and b). These pharmacological treatments did not alter the rates of OPC proliferation compared with control (Figures 2c and d). The results suggest that PKA activation is required for OPC migration mediated by IP receptor signaling.

Expression of prostacyclin and its receptor in the adult mouse CNS and MS patient brains. To test whether our in vitro observations reected the situation in vivo, we examined the role of prostacyclin in OPC accumulation in the LPC-induced demyelination model. Stereotaxic injection

of LPC into the dened adult spinal cord results in selective and focal myelin loss without producing signicant damage in adjacent cells and axons21,22 and induces subsequent

remyelination within 4 weeks.21 During the remyelination process, OPCs are recruited toward demyelinated lesions. We hypothesized that this recruitment is promoted by prostacyclin. To test this hypothesis, we rst investigated whether LPC injection into the spinal cord altered PGIS expression. In this model, we observed the complete ablation of resident OPCs at the lesion site 3 days after LPC injection (data not shown). Western blot analysis revealed that PGIS expression in the spinal cord tissue of LPC-injected mice increased compared with that in the control spinal cord tissue (Figure 3a). Immunohistochemical analysis demonstrated that majority of PDGFRa OPCs expressed IP receptor in the spinal cord (dorsal column; Figure 3b). Moreover, we detected IP receptor expression in PDGFRa cells in brain autopsy samples obtained from three individuals with

MS (Figure 3c).

Prostacyclin promotes OPC recruitment and remyelination. To determine whether prostacyclin has a crucial role in OPC recruitment and remyelination, we administered the IP receptor antagonist CAY10441 via osmotic mini-pumps with intrathecally placed catheters into the lesion site 3 days after LPC injection. We counted the number of PDGFRa/40,

6-diamidino-2-phenylindole (DAPI) double-positive OPCs in the dorsal column of the spinal cord 7 days after LPC injection (Figure 4a). The number of accumulated OPCs was signicantly attenuated in CAY10441-treated mice

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Figure 3 Prostacyclin and its receptor are expressed in the CNS tissue of mouse and MS patients. (a) Representative spinal cord sections immunostained for IP receptor (magenta) and PDGFRa (green) in control mice. Scale bar, 20 mm. (b) Representative brain section immunostained for IP receptor (green) and PDGFRa (magenta) in MS patients. Scale bar, 10 mm. (c) Western blot analyses for PGIS and a-tubulin expression in the spinal cord. Spinal cord tissues were obtained 4 days after LPC injection. PGIS level was normalized to that of a-tubulin. Values represent the meanS.E.M. (n 6). *Po0.05 compared with control

compared with vehicle-treated mice (Figures 4b and c). As these accumulated OPCs are considered to contribute to subsequent remyelination, we investigated whether the inhibition of prostacyclin signaling also prevented remyelination after LPC injection. We assessed the expression of MBP, a major component of CNS myelin, in the dorsal column of the spinal cord 14 days after LPC injection. Immunohistochemistry showed that the extent of MBP distribution in the spinal cord was lower in CAY10441-treated mice compared with control mice (Figures 4d and e). These results suggest that endogenous prostacyclin signaling is required for OPC recruitment and remyelination in the adult spinal cord.

We further examined whether prostacyclin can enhance OPC actions in the injured spinal cord. Intrathecal administration of iloprost facilitated OPC accumulation and myelin formation (Figures 4ce), suggesting that it has promising therapeutic potential owing to its remyelinating properties.

Prostacyclin improves motor function after demyelinating insult. Finally, we assessed whether prostacyclin promoted restoration of neurological function by examining

motor recovery after LPC-induced demyelination. Although focal demyelination in the dorsal column of the rodent spinal cord causes motor decits, this impairment is recovered spontaneously when the area is remyelinated.22,23 We used

two behavioral tests to evaluate impairment and subsequent recovery of hindlimb motor function: behavioral test recording (Figure 5a) and the basso mouse scale (BMS) (Figure 5b). Severe hindlimb impairment resulting from LPC injection was followed by spontaneous recovery up to day 28 after injection (Figures 5a and b). Consistent with the histological results, treatment with CAY10441 signicantly prevented spontaneous recovery of hindlimb function after LPC injection (Figures 5a and b), suggesting that endogenous prostacyclin contributes to the spontaneous recovery of motor function. We then investigated the therapeutic effect of prostacyclin on motor decits associated with demyelination and found that iloprost administration signicantly promoted the recovery of motor function. This signicant difference compared with vehicle control persisted from day 7 after LPC injection to day14. These results demonstrate that prostacyclin has therapeutic potential for treating neurological dysfunction that is the result of demyelination.

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Figure 4 Prostacyclin drives OPC accumulation and remyelination after LPC-induced demyelination in the spinal cord. (a) Representative spinal cord sections double-labeled for PDGFRa and DAPI. Arrowheads indicate OPCs, and the outlined arrowhead indicates a non-OPC in the dorsal funiculars of the spinal cord. Scale bar, 10 mm.

(b) Representative spinal cord sections showing PDGFRa-positive OPCs 7 days after LPC injection with or without IP receptor antagonist CAY10441 or iloprost treatment. Scale bars, 50 mm. (c) Quantication of accumulated OPCs in the spinal cord 7 days after LPC injection. Values represent the meanS.E.M. (n 5). (d) Representative

spinal cord sections showing MBP-positive myelin 14 days after LPC injection and treatment with CAY10441 and iloprost. Scale bar, 100 mm. (e) Quantication of the MBP-positive myelinated area in the spinal cord 14 days after LPC injection. Values represent the meanS.E.M. (n 5). *Po0.05 and **Po0.01 compared with control

Prostacyclin has been reported to be an inammatory mediator that is associated with cytokine expression24,25 and

inammatory cell function.26 To test whether neurological recovery is independent of inammation, we measured cytokine expression in spinal cord tissue treated with each pharmacological reagent. There were no differences in the amounts of the following cytokines: interleukin (IL)-1a, IL-1b,

IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, interferon-g,

tumor necrosis factor-a, monocyte chemoattractant protein-1, granulocytemonocyte colony-stimulating factor, macrophage colony-stimulating factor, and regulated upon activation normal T cell expressed and secreted released in the spinal cord tissue obtained from control and iloprost- or CAY10441-treated mice 4 days after LPC injection (data not shown). These data suggest that prostacyclin signaling did not affect inammation at the demyelinating lesions.

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Figure 5 Prostacyclin promotes recovery from motor decits caused by LPC-induced spinal cord demyelination. (a and b) The graphs depict the beam walking score (a) and BMS score (b) to estimate motor function. Values represent the meanS.E.M. (control: n 9, sham: n 11, iloprost: n 7, and CAY10441:

n 7). *Po0.05 and **Po0.01 compared with control

Days Post Operation

Taken together, our ndings show that prostacyclin drives remyelination through OPC accumulation by activating IP receptor signaling. This effect appears to enhance hindlimb motor function recovery following localized demyelination induced by LPC injection.

Discussion

In this study, we elucidated the role of prostacyclin signaling in remyelination both in vitro and in vivo. The results of our in vitro migration assays demonstrate that prostacyclin promotes OPC migration via PKA signaling. In the commonly used model of LPC-induced demyelination of the dorsal column of the spinal cord, PGIS expression was upregulated. Treatment with IP receptor antagonist prevented spontaneous OPC accumulation and remyelination in the lesion after LPC injection. Conversely, prostacyclin analog enhanced OPC accumulation and remyelination. Finally, two behavioral analyses identied prostacyclin as the factor responsible for enhancing neurological recovery after demyelination.

The involvement of prostaglandins in MS pathogenesis was suggested by the nding that some prostaglandins were increased in the cerebrospinal uid of MS patients.27

It is generally accepted that MS is an autoimmune disease characterized by immune activation and cell inltration.

The role of prostaglandins in MS has been mainly studied with a focus on immunoregulation using the prevailing animal model of MS, experimental autoimmune encephalomyelitis (EAE).28 Cytosolic phospholipase A2a (cPLA2a) is one of the critical enzymes involved in generating multiple prostaglandins. cPLA2a selectively cleaves arachidonyl phospholipids to release free arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase pathway. It is reported that cPLA2a-decient mice, which cannot produce endogenous levels of PGs, are resistant to EAE induction.29 In addition, adoptive transfer experiments revealed that cPLA2a is also involved in both the induction and the effector phases of EAE.29

There is ample evidence to suggest that cPLA2a and its products are related to inammation and autoimmune disease through PG production. This is in agreement with recent reports suggesting that PGs, especially PGE2, has a pro-inammatory role in EAE pathogenesis.30 Microsomal prostaglandin E synthase-1-decient mice show less severe EAE symptoms and lower IL-17 and interferon-g production compared with control mice.31 Thus, PGs are considered to enhance inammation and EAE severity. However, it is important to note that PGs may have other role(s) besides as an inammatory factor. Thromboxane A2, another arachidonic acid metabolite, is known to promote oligodendrocyte proliferation and survival.32 Oligodendrocytes are considered to contribute to spontaneous myelin repair, which is a characteristic feature in some acute MS lesions.4,5 In our study, prostacyclin

facilitated spontaneous OPC recruitment, and this effect was independent of cytokine expression. This supports a previous report that prostacyclin fails to ameliorate EAE progression.25

Previous studies have reported that cAMP elevation enhances remyelination in the adult CNS.33,34 Relevant to remyelination, the adult OPCs undergo several developmental steps to become mature cells that initiate remyelination. Several studies have reported that cAMP analogs have been shown to induce the expression of myelin constituents in cultured oligodendrocytes,35,36 indicating that cAMP elevation induces OPC differentiation. On the other hand, OPC proliferation is not affected by cAMP level.37,38 PKA, the major

target for cAMP action, is generally considered to induce the hallmarks of cell migration (e.g., actin lament assembly and activation of small G proteins).20 Indeed, our in vitro experiments showed that OPC migration upregulated by prostacyclin is dependent on PKA activation.

IP receptor is expressed in various mouse organs, including thymus, spleen, brain, and vascular tissues.14 The expression pattern is inferred to be critical for the development of the cardiovascular, immune, and nervous systems. However, IP receptor-decient mice are viable, reproductive, and normotensive. Furthermore, no morphological or histological abnormalities were detected.39 Meanwhile, IP receptor-decient mice showed slightly altered responses to acute nociceptive stimuli,39 suggesting that the role of endogenous prostacyclin-IP receptor signaling is restricted to pathological conditions. Indeed, we revealed that abundant prostacyclin expression in demyelinating lesion contributes to OPC recruitment through IP receptors, which is constitutively expressed on OPCs. Thus, lack of endogenous prostacyclin-IP receptor signaling might be compensated during the developmental stage and in physiological conditions, but

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endogenous prostacyclin-IP receptor signaling may be required for the neural network restoration under some pathological conditions.

Which cells produce prostacyclin in the diseased CNS? Angiogenesis, the formation of new blood vessels from an existing capillary, is concurrent with tissue repair and is essential for wound healing in adults. Prostacyclin is synthesized by vascular endothelial cells and smooth muscle cells. We recently reported that PGIS expression was increased in vascular endothelial cells in EAE mice.18 Therefore, vascular endothelial cells may be candidate cells for prostacyclin production.

Emerging data indicate that cellcell interactions between the endothelium and neuronal cells helps sustain brain homeostasis and function.4042 It is now well accepted that

this can be partially explained by the ability of endothelial cells to produce trophic factors. Recent studies have shown that endothelium-derived factor(s) also act on OPCs, inducing their proliferation in vitro.43 Our ndings support the hypothesis that an oligovascular niche might also exist in the adult CNS and is partially regulated by prostacyclin. Importantly, this oligovascular niche may provide a promising new approach for treating demyelinating diseases.

Materials and MethodsMice. C57BL/6 J mice were obtained from SLC Japan (Hamamatsu, Japan). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Osaka University.

Primary culture of OPCs. OPCs were prepared from whole brains obtained from postnatal day 1 mice. We dissociated fresh mouse brain in Dulbeccos modied Eagles medium (DMEM, Invitrogen, Carlsbad, CA, USA). After washing, we ltered the cell suspension through a 70-mm cell strainer to obtain a single-cell suspension. Cells were incubated with A2B5-specic antibody-coated magnetic beads (Miltenyi-Biotec, Bergisch Gladbach, Germany) to isolate selectively OPCs. The isolated cells were plated a density of 4 104 cells/well into poly-L-lysine-

coated four-well chamber slides (Nunc, Rochester, NY, USA) in DMEM containing 4 mM L-glutamine (MP Biomedicals, Aurora, OH, USA), 1 mM sodium pyruvate (Sigma, St.Louis, MO, USA), 0.1% bovine serum albumin (BSA, Sigma), 50 mg/ml apo-transferrin (Sigma), 5 mg/ml insulin (Sigma), 30 nM sodium selenite (Sigma), 10 nM biotin (Sigma), 10 nM hydrocortisone (Sigma), 10 ng/ml platelet-derived growth factor (PDGF)-AA (PeproTech, Rocky Hill, NJ, USA), and 10 ng/ml basic-broblast growth factor (FGF) (PeproTech). Cells were maintained at 37 1C and at 5% CO2.

After culturing for 24 h, the cells were xed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 min at room temperature. The cells were then permeabilized in PBS containing 5% BSA and 0.1% Triton X-100 for 1 h at room temperature. Cells were double-labeled with mouse anti-A2B5 (1 : 300, Millipore, Bedford, MA, USA) and rabbit anti-IP receptor (1 : 100, Cayman, Ann Arbor, MI, USA) diluted in PBS containing 5% BSA and 0.1% Triton X-100 overnight at 4 1C. The cells were visualized with secondary Alexa Fluor 488- or 568-conjugated goat antibody to rabbit IgG and goat antibody to mouse IgM (1 : 500,

Invitrogen). Images were acquired using an upright microscope and DP-controller image system (Olympus, Tokyo, Japan).

Primary culture of cortical neurons. Cortical neurons were prepared from cerebral cortices obtained from postnatal day 1 mice. The cerebral cortices were dissociated by trypsinization (treatment with 0.25% trypsin (Invitrogen) and0.5 mg/ml DNase (Sigma) in PBS for 15 min at 37 1C) followed by resuspension in DMEM containing 10% fetal bovine serum (FBS, Invitrogen). The isolated cells were plated on poly-L-lysine-coated dishes at a density of 4 104 cells/well and

maintained at 37 1C and at 5% CO2.

Migration assay. OPC migration was examined in transwell cell-culture inserts (6.5-mm diameter, 8-mm pore size, Corning Costar, Corning, NY, USA).

Cells were suspended in 10% FBSDMEM at a concentration of 4 104 cells/ml.

A 0.1 ml cell suspension (nal concentration: 4000 cells/well) was added to the upper compartment, and cells were then incubated for 16 h. After xation with 4% PFA, the transwell lters were incubated for 30 min in a solution of 4,6-diamidinophenylindole (DAPI, Santa Cruz, Santa Cruz, CA, USA) at the nal concentration of 1 mg/ml in PBS. To avoid the observation of non-migrating OPCs, cells on the upper surface of the lter were removed by wiping with lter paper. Images of the bottom of the transwell lter were captured with an upright microscope using a 4 objective

(Olympus). We counted the number of DAPI cells and calculated the ratio of cultured cells treated with pharmacological agents to the number observed under control conditions. For pharmacological experiments, the following reagents were added into the lower compartment at the beginning of the culture period: Sp-cAMPS (PKA agonist, Sigma), Rp-cAMPS (PKA antagonist, Sigma), iloprost (a stable prostacyclin analog, Cayman Chemical), and cicaprost (another prostacyclin analog, Cayman Chemical).

Proliferation assay. OPCs were plated at a density of 8 104 cells/ml in

DMEM containing 10% FBS with the indicated agents for 16 h. Cell proliferation was estimated by counting the A2B5/DAPI double-positive cells. Images were captured with an upright microscope equipped with a 4 objective (Olympus).

We calculated a ratio of cultured cells treated with pharmacological agents to the number observed under control conditions.

Western blotting. Cells or spinal cord tissues were lysed with RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris at pH 7.4) containing protease inhibitors (complete protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany). Cells or tissue lysates were then homogenized and centrifuged at 17 400 g for 20 min at

4 1C, and protein concentrations in the supernatants were determined with bicinchoninic acid protein assay (Thermo Scientic, Waltham, MA, USA). Proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene diuoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween-20, the membranes were incubated with solutions of rabbit anti-IP receptor (1 : 500, Cayman Chemical), mouse anti-a-tubulin (1 : 500, Santa Cruz), or rabbit anti-PGIS (1 : 100; Cayman

Chemical) overnight at 4 1C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 5000, Cell Signaling Technology,

Danvers, MA, USA) for 1 h at room temperature. Signals were detected by an enhanced chemiluminescence (ECL) system (GE Healthcare, Munich, Germany), and protein expression quantication was performed using ImageJ software (NIH; http://rsb.info.nih.gov/ij/

Web End =http://rsb.info.nih.gov/ij/ ).

LPC-induced demyelination in mouse spinal cord. Adult female mice were anesthetized with sodium pentobarbital before undergoing laminectomy at the thoracic level 11-12. We injected 2 ml 1% LPC (Sigma) dissolved in saline into the dorsal column midline at a depth of 0.5 mm.

For administration of pharmacological reagents, osmotic pump (model no. 1002 and no. 1007D; ALZET Corp., Cupertino, CA, USA) was lled with vehicle solution (saline), CAY10441 (IP receptor antagonist, 20 mg/kg/day; Cayman Chemical), or iloprost (20 mg/kg/day, Cayman Chemical). The delivery tube connected to an osmotic pump was placed close to the lesion 3 days after LPC injection. The pump was implanted subcutaneously in the dorsal skin.

Immunohistochemistry of mouse tissue. Mice were transcardially perfused with 4% PFA in PBS. The spinal cords were post-xed with 4% PFA in PBS for 24 h and then transferred sequentially into 20 and 30% sucrose solution until they were equilibrated. The spinal cords were sectioned in the transverse plane on a sliding microtome at 20 mm and were mounted on Matsunami adhesive silane-coated Superfrost/Plus slides (Matsunami, Osaka, Japan). Five to seven sections were obtained from every 100 mm that included the lesion epicenter.

For immunohistochemistry, sections were treated with 5% BSA plus 0.1% Triton X-100 in PBS for 1 h at room temperature. The sections were incubated with primary antibodies diluted in PBS containing 5% BSA and 0.1% Triton X-100 overnight at 4 1C, followed by incubation with secondary antibodies for 1 h at room temperature.

Images were taken by a uorescence (Olympus BX51, DP71) or confocal laser-scanning microscope (Olympus FluoView FV1000). The following primary antibodies were used: rat anti-CD140a (PDGFRa, 1 : 50; BD Biosciences,

Erembodegem, Belgium), rabbit anti-IP receptor (1 : 100, Cayman Chemical), rat anti-MBP (1 : 50, Abcam, Cambridge, MA, USA). Secondary antibodies were Alexa

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Fluor 488- and 568-conjugated antibodies produced in goat (1 : 500, Invitrogen). Nuclear staining was performed with DAPI.

To assess OPC accumulation in the spinal cord, we counted the number of PDGFRa/DAPI double-positive cells in the dorsal column of spinal cord. We obtained the cell number of 10 areas (50 50 20 mm) from 100-mm intervals in

the dorsal column of the spinal cord. For quantication of myelin formation, we measured the MBP-positive area in the dorsal column of the spinal cord.

Immunohistochemistry of human tissues. We obtained autopsied spinal cord tissue from three individuals with relapse-remitting MS. We xed samples with formalin and then embedded the tissue in parafn before preparing 4-mm-thick slices for immunohistochemistry. We deparafnized sections with xylene followed by 100% ethanol. Antigen retrieval was performed by incubation at 98 1C for 40 min in citrate buffer (10 mM, pH 6.0). We incubated tissue samples with primary antibodies specic for human PDGFRa (1:100, R&D Systems,

Minneapolis, MN, USA) and IP receptor (1 : 100; Cayman Chemical). We used Alexa Fluor 488- and 568-conjugated antibodies (1 : 500, Invitrogen) for secondary antibodies. The research protocol was approved by the Human Use Review Committees of the Graduate School of Medicine, Osaka University, and Toneyama National Hospital, for the Protection of Human Subjects. Informed consent was obtained from all subjects.

Basso mouse scale. Recovery of hindlimb motor function was scored by the BMS open-eld locomotor rating scale, which was developed specically for mice.44 The score ranges from 0 (complete paralysis) to 9 (normal mobility), and the number of errors at each footstep was measured for 100 steps. Mice were observed individually for 2 min in an open eld.

Behavioral data recording. Behavioral scoring was carried out while viewing the recording in slow motion on a standard video recorder.23 Scores were given to every step according to the owing scheme: 0, normal step; 1, a minor error (slight insecurity of foot placement); or 2, major error (foot slipped completely from the beam surface). The total score for each step per mouse was used for subsequent statistical analysis.

Cytokine analysis. Spinal cord tissues at the level of Th11-12 were homogenized in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA containing protease inhibitors. Samples were centrifuged at 17 400 g for 10 min at 4 1C, and the supernatants were collected. The

expression proles of a panel of cytokines were measured using the Q-Plex Mouse Cytokine Array (Quansys Biosciences, Logan, UT, USA) according to the manufacturers instructions.

Statistical analysis. Data are presented as meanS.E.M. For the analysis of PGIS protein expression, differences between groups were examined using Students t-tests. For behavioral analysis, differences between groups were examined using two-way repeated measure analyses of variance (ANOVAs) followed by post hoc Bonferroni tests. Other experiments were analyzed with one-way ANOVAs followed by post hoc TukeyKramer tests.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. This work was supported by a Grant-in-Aid for Scientic Research on Innovative Areas (25122711) from the Japan Society for the Promotion of Sciences to RM, a Grant-in-Aid from Ministry of Health, Labour and Welfare to RM, the Osaka University Program for the Support of Networking among Present and Future Researchers to RM, and a Grant-in-Aid for Scientic Research (S) from JSPS (25221309) to TY.

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