Molecular and Cellular Neuroscience 42 (2009) 288–295

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Molecular and Cellular Neuroscience

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Role of plasminogen activation in neuronal organization and survival

Benoît Ho-Tin-Noé a,1, Hervé Enslen b,c,d,1, Loïc Doeuvre e,f, Jean-Marc Corsi b,c,d,H. Roger Lijnen g, Eduardo Anglés-Cano e,f,⁎a Inserm U698, Université Paris Diderot-Paris 7, 75018 Paris, Franceb Inserm U839, 75005 Paris, Francec Université Pierre et Marie Curie, 75005 Paris, Franced Institut du Fer-à-Moulin, 75005 Paris, Francee Inserm U919, 14074, Université de Caen, Caen, Francef CNRS UMR6232, 14074, Université de Caen, Caen, Franceg Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium

⁎ Corresponding author. Inserm U919 Serine ProteasGIP Cyceron/Bd Henri Becquerel, 14074-cedex, Caen, Fr

E-mail address: Eduardo.Angles-Cano@inserm.fr (E.1 These authors contributed equally to this work.

1044-7431/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.mcn.2009.08.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 April 2009Revised 5 July 2009Accepted 4 August 2009Available online 14 August 2009

We characterized the interactions between plasminogen and neurons and investigated the associated effectson extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding ofplasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA)degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from thematrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysineresidues and the proteolytic activity of plasmin. Surprisingly, 24 h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinasephosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting thatprolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival byplasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/orextravasate during vascular accidents or inflammatory processes.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Extracellular matrix (ECM) remodeling and cell migration in thevessel wall and the brain are regulated by two major proteolyticsystems: the plasminogen activation system and the matrix metallo-proteinases (MMPs) (Lijnen, 2002; Lo et al., 2002). In vessels, tissue-type plasminogen activator (tPA) generates plasmin on fibrin clotsleading to fibrinolysis (Wiman and Collen, 1978), whereas theurokinase-type PA (uPA) is mainly involved in vascular remodelingand cell migration (Plow and Hoover-Plow, 2004). In the CNS, tPA isabundantly expressed (Sappino et al., 1993) and acts on plasminogen-dependent non-fibrin substrates (Tsirka et al., 1997) and onplasminogen-independent targets such as the NMDA (N-Methyl-D-Aspartate) receptor (Nicole et al., 2001). These activities have beenimplicated in hippocampal function, neurite outgrowth and pathfind-ing during brain development and plasticity (Seeds et al., 1997),and also participate in excitotoxic-mediated neuronal cell death by

es in Neurovascular Pathology,ance. Fax: +33 2 31470222.Anglés-Cano).

ll rights reserved.

degrading laminin, a component of the ECM (Chen and Strickland,1997). We have previously shown that degradation of ECM proteins,including laminin and fibronectin, bymembrane-bound plasmin leadsto detachment and apoptosis of vascular cells (Meilhac et al., 2003), aphenomenon that may be of relevance in pathological situations invivo (Rossignol et al., 2006). To explore the possibility that a similarphenomenon may occur on mouse cortical neurons, we characterizedtheir capacity to activate plasminogen as well as its effects on ECM,signaling transduction pathways, morphology, adhesion and survivalof neurons. Our results show that neurons activate plasminogen in atPA-dependent manner, and that the subsequent plasmin formationleads to degradation of the ECM and neuron detachment. Surprisingly,plasmin-detached neurons form cellular clusters associated withstable focal adhesion kinase (FAK) signaling and survival, unlessplasmin(ogen) exposure was extensively prolonged.

Results

Plasminogen binds to mouse cortical neuron and is activated by tPA onneuron surface

Plasminogen bound to cortical neurons in a saturable (Fig. 1A,Kd=0.45±0.06 µM), specific and lysine-dependent manner (Fig. 1B).

Fig. 1. Plasminogen binds to cortical neurons in a lysine-dependent manner. A. Bindingof plasminogen to mouse cortical neurons (Kd=0.45±0.06 µM) was detected using amAb to plasminogen. B. Effect of ε-ACA (solid line) and carboxypeptidase B (CpB,dashed-line) on plasminogen binding to cortical neurons. Results are expressed as apercentage (mean±SD, n=3) relative to the amount of plasminogen bound toneurons in the absence of ε-ACA or CpB treatment.

Fig. 2. Activation of plasminogen on neurons via tPA. A. Effect of tPA-blocking IgG ( )and uPA-blocking IgG (⋄) on plasmin formation onto neurons as measured with achromogenic substrate. Results are expressed as a percentage (mean±SD, n=3)relative to plasmin formation in the presence of non-immune IgG. Inset. Neuron lysates(NL) and purified human tPA as reference were analyzed by either western blotting(left panel) using a polyclonal antibody to mouse tPA and by fibrin-agar zymography(middle panels); right panel: fibrin plate assay for tPA activity in neuron lysates and itsinhibition by the tPA-blocking IgG. B. Kinetics of plasminogen activation onto neurons(Km=45.7±11 nM) was followed by detecting plasmin activity with a colorimetricassay. Inset. Effect of ε-ACA on plasminogen activation by neurons, IC50=1.2 mM.Western blot analysis of cell-conditioned media using a mAb to plasmin(ogen). Lane 1:control plasminogen (Pg), lane 2: control plasmin (Pn), lane 3: untreated-controlneurons, lane 4 to 7: neurons treated respectively with 0.25, 0.5, 1 and 2 µMplasminogen. C. Effect of tPA-blocking IgG and uPA-blocking IgG on plasminogenactivation as measured by western blotting of neuron culture supernatants. Pg, Pn,migration of reference plasminogen and plasmin. Ab: antibody. Concentrations used:Pg: 0.5 µM, IgG: 40 µg/ml, ε-ACA: 25 mM.

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Plasminogen binding to neurons was indeed prevented whencarboxy-terminal lysines on cells were cleavedwith carboxypeptidaseB or when lysine-binding sites of plasminogen were blocked with thelysine analogue ε-ACA (Fig. 1B).

Analysis of neuron lysates by Western blot and zymographyallowed identification of tPA (Fig. 2A, inset). The selective inhibition ofplasminogen activation by an anti-mouse tPA IgG but not by an anti-mouse uPA IgG (Fig. 2A) further confirmed that the activator ofplasminogen produced by mouse cortical neurons is tPA. Inconsequence, incubation of plasminogen with mouse cortical neuronsresulted in its conversion to plasmin (Fig. 2B, Western blot inset) in adose-dependent and saturable manner (Km=45.7±11 nM). Plas-minogen activation was specifically inhibited by ε-ACA (Fig. 2B, insetgraph, IC50=1.2 mM; Fig. 2C, right lane), thus indicating thatplasminogen binding to neurons is required for its activation.Cleavage of plasminogen by tPA was further confirmed by its specificinhibition with a blocking antibody to mouse tPA (Fig. 2C). Verysimilar results were obtained in terms of plasmin generation whenhuman plasminogen was substituted with murine plasminogen (notshown). Finally, absence of glial participation was evidenced bysimilar plasmin formation rates in the presence of the proliferationinhibitor Ara-C (Table 1). Altogether, this data indicate that molecularassembly of tPA and plasminogen onto mouse cortical neurons isrequired for in situ plasmin formation.

Plasmin abrogates neuron–substratum interactions but notneuron–neuron contacts

Incubating neurons with increasing concentrations of plasmino-gen resulted in a dose-dependent retraction of the cell layer (Fig. 3A).Microscopic observation of the retracted cell layer revealed thatplasminogen-treated neurons were reorganized into compact clustersinterconnected by fasciculating fibers (Fig. 3B). Quantification of cell

Table 1Occurrence of neuron retraction and detachment in various conditions.

Pg Pg/Ara-C

Pg/ε-ACA

Pg/α2-AP

Pg/PP2

rPg-S741A Pn Pn/VPK

CM CM/Aprot

+ + − − + − + − + −

Neuron retraction and detachment were visually assessed under a light microscope theday following stimulation with either 0.5 µM plasminogen (Pg) supplemented or notwith 3 µM Ara-C (Pg/Ara-C), 10 mM ε-ACA (Pg/ε-ACA), 1 µM α2-AP (Pg/α2-AP) or10 µM PP2 (Pg/PP2); 0.5 µM plasminogen active site mutant (rPg-S741A); 0.5 µMplasmin (Pn) supplemented or not with 10 µM VPK (Pn/VPK); conditioned mediumfrom neurons stimulated with 0.5 µM plasminogen for 18 h (CM) supplemented or notwith 1 µM aprotinin (CM/Aprot).

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adhesion revealed that neuron clusters were loosely attached to thesubstratum as they were easily flushed away by washing (Fig. 4A).Loss of adhesion of the neuron clusters to the ECM in responseto plasmin was associated with degradation of laminin, fibronectinand tenascin-C (Fig. 4C). This suggests that neuron detachment andconcomitant clustering involve proteolytic degradation of the ECM byplasmin. The addition of either ε-ACA (Figs. 3A, 4A and Table 1) thatinhibits plasminogen binding and activation, or α2-AP (Fig. 4B andTable 1) that inhibits plasmin activity, prevented retraction of the cellmonolayer and loss of adhesion indicating that both the plasminogen-binding domains and plasmin activity are required for the detectedeffects.

Fig. 3. Plasminogen activation induces retraction and clustering of mouse corticalneurons. A. Retraction of neuron monolayers as a function of the concentration ofplasminogen added, measured at 24 h of incubation. The retraction index wascalculated as the ratio of control to plasminogen-treated MTT-stained neuronal surfacearea. Inset. Scan of the corresponding MTT-stained wells showing intact and retractedneuron monolayers. B. Phase-contrast micrographs (50×) of control and plasminogen(0.5 µM)-treated cortical neurons after 24 h of incubation. Arrows indicate multicel-lular clusters (black arrow) interconnected by neurite bundles (white arrow).

To determine if the observed changes in neuron organizationwere a consequence of proteolysis-induced cell detachment by theplasmin catalytic domain or an independent and specific response toprotein-binding domains of plasmin(ogen), we incubated the cellswith either a recombinant active site mutant of plasminogen or withactive site-blocked plasmin. Neither the plasminogen active sitemutant rPg-S741A (Fig. 4A and Table 1) nor active site-blockedplasmin (Table 1) could induce cell detachment and aggregation.In contrast, transfer of conditioned media from plasminogen-treatedneurons to fresh cells resulted in detachment and aggregation ofthe fresh neurons that were efficiently prevented by aprotinin, aninhibitor of plasmin (Table 1 and Suppl. Fig. 1). Altogether, these dataindicate that plasmin with an active site is required to elicit neurondetachment and aggregation.

Neurons maintain stable levels of FAK phosphorylation uponplasmin-induced detachment and clustering

The formation of neuronal clusters upon plasmin-induced detach-ment is a strikingly unique and different response to plasmin forma-tion by cellular tPA as compared to other adherent cell types suchas CHO-K1 cells that are detached and fully dissociated by plasmin(Meilhac et al., 2003; Rossignol et al., 2004). Cell–cell and cell–ECMinteractions are tightly intertwined with intracellular signalinginvolved in the regulation of cell morphology and survival. To inves-tigate whether formation of neurons clusters in response to plas-min was associated with specific regulation of signaling pathwaysdifferent from those commonly associated with loss of adhesion, suchas dephosphorylation (inactivation) of FAK (Alahari et al., 2002), wemonitored in plasmin-detached neurons, the activation of proteinkinases known to be implicated in anchorage-dependent signaling.During the first hour following the addition of plasminogen to corticalneurons, no morphological changes or effects on the level of phos-phorylation of FAK, Src family members (SFK) and Akt were observedcompared to untreated neurons (data not shown). Furthermore,despite detachment and progressive clustering of the neurons overthe next 17 h, the activation/phosphorylation of FAK, SFK and Aktwere still unchanged compared to control neurons (SupplementaryFig. 2). However, 24 h after the addition of plasminogen, the acti-vation of SFK was promoted in detached clusters of cortical neuronsalthough both Akt and FAK phosphorylation remained unaffected(Fig. 5A). These results contrast with those observed in CHO cells,which were fully detached and dissociated 24 h after the addition ofplasminogen (Fig. 5B) and showed a dramatic dephosphorylation ofFAK (Fig. 5B). Thus, although similar effects of plasminogen on Aktand Src were observed in detached clustered neurons and dissociatedCHO-K1 cells, neurons in clusters maintain stable levels of phosphor-ylated FAK despite plasmin-induced loss of adhesion to the ECM.

Prolonged exposure of neurons to plasminogen decreases neuronviability

Plasminogen activation-induced cell detachment was previouslyshown to cause apoptosis of endothelial cells, vascular smoothmusclecells and CHO-K1 cells (Meilhac et al., 2003; Reijerkerk et al., 2003;Rossignol et al., 2004). No difference in TUNEL index betweenplasminogen-treated and control neurons could be detected after24 h of incubation (not shown). However, the TUNEL index ofplasminogen-treated neurons was of 9.2±3.8% at 36h of incubation(as compared to 2.3±1.4% in the control), and increased to 24.4±2.6% after 72h of incubation (compared to 2.9±0.9% for untreatedneurons) (Figs. 6A and B). Neurons whose nuclei appeared clearlyfragmented and condensed in DAPI staining and TUNEL also displayedpositive immunostaining for activated caspase-3 (Fig. 6C), confirmingthe induction of apoptosis, which was associated with partialdegradation of FAK, Src and Akt (Fig. 6D). Of note that residual non-

Fig. 4. Plasmin-induced degradation of extracellular matrix proteins leads to loss of neuron adhesion. A. Effect on the adhesion of cortical neurons of a 12-h incubation with eitheractive site plasminogen mutant (rPg) or wild-type plasminogen determined in the presence or absence of ε-ACA by the colorimetric MTT test. Results are expressed as a percentage(mean±SD, n=3) relative to the amount of adherent untreated-control cells. *, pb0.0001 versus control. B. Effect of α2-AP on neuron detachment induced by a 24-h incubationwith 0.5 µM plasminogen. C. Western blots of cortical neurons-conditioned media after 24 h of incubation with plasminogen. Membranes were probed with IgG to fibronectin,laminin or tenascin. The relative molecular mass of marker proteins is indicated on the left.

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degraded FAK, SFK and Akt remained phosphorylated (data notshown). As for plasmin-induced neuron detachment, the effects ofplasminogen activation on neuron viability were prevented byaprotinin, thus indicating a requirement for plasmin activity (notshown). These results indicate that long-term exposure (N24 h) toplasminogen may cause apoptosis of neurons.

Discussion

In this study, we report a hitherto undescribed phenomenon,the re-organization of neurons into compact clusters in response toplasminogen exposure. We show that this process is initiated byplasminogen binding and activation onto neurons that expresstPA (Figs. 1 and 2). The plasmin formed in situ degrades ECM pro-teins such as laminin, fibronectin and tenascin-C leading to loss ofcell adhesion and concomitant formation of neuronal clusters (Figs. 3and 4). Prevention of this sequence of events by ε-ACA,α2-antiplasminor the plasminogen active site mutant rPg-S741A, indicates that bothplasminogen-binding sites and plasminwith an active site are required.We further report that conditioned media from plasminogen-treatedneurons induces neuron detachment and aggregationwhen transferredto fresh neurons. Thiswas however prevented by aprotinin, an inhibitorof plasmin. These results confirm that plasmin-mediated proteolysis isrequired to elicit neuron detachment and clustering and exclude aneffect of soluble mediators released by neuron or from the ECM uponplasmin formation.

The clustering effect of plasmin on neurons contrasts with thepreviously described plasmin-induced dissociation of other adherent

cells capable of tPA-mediated plasminogen activation such as vascularsmooth muscle cells, endothelial cells or CHO-K1 cells (Meilhac et al.,2003; Reijerkerk et al., 2003; Zhang et al., 2003; Rossignol et al., 2004).Neuron organization has been shown to be highly dependent on thenature of the substratum or medium components (Kingsbury et al.,1985; Monnerie et al., 1996; Maltais et al., 2003). Our results indicatethat remodeling of the substratum by plasmin represents a potentialmechanism for the regulation of neuron organization. As both tPA andplasminogen are expressed in or may reach the CNS (Sappino et al.,1993; Tsirka et al., 1997), the potent clustering effect of plasmin onneurons suggests that plasminogen activation may participate innervous tissue organization in general and neuronal development inparticular. Functional neuron networks including adhesive contactsbetween neural cell bodies to form histological cellular clustering(Becker and Redies, 2003; Price et al., 2002), and fasciculation of fibers(Kunz et al., 1998; Treubert-Zimmermann et al., 2002) has beenindeed previously reported.

Our results suggest that the drastic effect of plasmin on neuronorganization affect cell–cell and cell–ECM contact-dependent signal-ing pathways. The uncommon clustering response of neurons toplasminogen is associated with effects on signaling pathways, some ofwhich are similar to the ones induced in cells that are dissociatedby plasmin. No differences were indeed found between detachedclustered neurons and dissociated CHO-K1 cells in the effects ofplasminogen on Akt and Src during the first 24h after its addition(Fig. 5, Suppl. Fig. 2), suggesting that these kinases may not beresponsible for the distinct cell phenotypes observed in response toplasmin formation. Furthermore, preincubation of plasminogen-

Fig. 5. Regulation of protein kinases in response to plasmin formation on CHO-K1 cells and cortical neurons. Western blot analysis of phospho-Tyr-397-FAK (P-FAK), phospho-SFK(P-SFK), phosphor-Ser-473-Akt (P-Akt) and total levels of FAK, Fyn and Akt were performed in cortical neurons (A) and CHO-K1 cells (B) following 24 h incubation with 0.5 µMplasminogen (Pg). Representative photographs of control and plasminogen-treated cells are shown on the top of each panel. In response to Pg treatment, neurons detach as clusterswhereas CHO-K1 cells detach and dissociate. Bar graphs represent the fraction of phosphorylated-active enzyme (Phospho-active form/total) in plasminogen-treated cells,expressed as a percentage+/−sem of the ratio of phosphorylated-active enzyme (Phospho-active form/ total) in the control cells, set to 100%+/−sem (n=5–6).

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treated neurons with PP2, a pharmacological inhibitor that preventedSFK activation did not affect neuronal clustering (Table 1), thusconfirming that SFK is not involved in this phenomenon. Loss of celladhesion is commonly associated with apoptosis (Meredith et al.,1993) and loss of FAK phosphorylation (Alahari et al., 2002), as shownin this study for CHO-K1 cells (Fig. 5B). In contrast, the level of FAKphosphorylation in neurons was unaffected by plasmin-induceddetachment (Fig. 5A) and no significant apoptosis was observed at24 h. Thus, neurons displayed surprising signaling features bymaintaining FAK phosphorylation and survival despite plasmin-induced loss of adhesion to the substratum. It is noteworthy thatthe level of FAK phosphorylation in neurons remained stable inparallel with their evolving clustering status (Supplementary Fig. 2and Fig. 5A). This suggests that cell–cell contacts in the neuronalclusters may maintain phosphorylation of FAK and compensatethereby FAK dephosphorylation from loosing cell–ECM contacts.

A constitutively active form of FAK was previously reported toprotect normal cells against anoïkis (Frisch et al., 1996). The persistenceof FAK phosphorylation in neurons, upon loss of adhesionmay promotetheir resistance to cell detachment-induced apoptosis and explain theirstable survival at 24 h following plasminogen addition. However,the exact role of FAK in the phenotypic changes observed in neuronsin response to plasminogen remains to be established.

Interestingly, while neuron viability and the phosphorylation levelof FAK in neurons remained stable during the first 24 h (Fig. 5, Suppl.Fig. 2 and data not shown), prolonged exposure (72 h) to plasmin(ogen) resulted in an increasing rate in neuron apoptosis associatedwith a decrease in the total amount of FAK and other signallingproteins (Fig. 6). As for plasminogen-induced neuron clustering,plasminogen-induced neuron death was prevented by aprotinin,indicating that it relied on plasmin formation and activity. Thissuggests that prolonged exposure of neurons to plasminogen maycause neurotoxicity in the presence of tPA. tPA and plasminogenproduced in the hippocampus were previously shown to sensitizeneurons to cell death via the degradation of laminin, in a model of

excitotoxicity induced by intrahippocampal injections of kainate(Chen et al., 1999; Chen and Strickland, 1997). Our data indicate thatprolonged exposure of neurons to plasmin may be neurotoxicindependently of kainate stimulation. It is conceivable that neuronsmay be in contact with locally produced plasminogen (Basham andSeeds, 2001) or in case of disruption of the blood–brain barrier. Recentfindings indeed showed that large openings in blood–brain barrieroccur following reperfusion after transient focal cerebral ischemia andallow extravasation of plasma proteins through cerebral vessels(Nagaraja et al., 2008). Breakdown of the blood–brain barrier andsubsequent plasminogen leakage into the brain may contribute toneuron death in plasminogen-nonproducing areas. Reperfusion injuryfollowing ischemic stroke can indeed affect brain areas others thanthe hippocampus.

In conclusion, our results indicate that exposure of tPA-expressingneurons to plasminogen induces a biphasic neuron responsemediated by plasmin formation. While incubation with plasminogenfirst evokes clustering of neurons that maintain stable FAK signalingand survival despite loss of adhesion to the substratum, persistentexposure to plasmin results in decreased neuron viability. Theclustering consequences of plasmin on neurons suggest that limitedplasminogen activationmay contribute to nervous tissue organizationduring development, while uncontrolled plasmin formation may beneurotoxic. Plasmin effects on both neuronal organization andsurvival may be of pathophysiological relevance as plasminogen isexpressed by microglial cells (Nakajima et al., 1992) or mayextravasate into the brain during vascular accidents or inflammatoryprocesses.

Experimental methods

Materials

Native human plasminogen, recombinant mutant human plasmin-ogen (rPg-S741A), plasmin and α2-antiplasmin, murine plasminogen

Fig. 6. Long-term exposure of neurons to plasminogen decreases neuron viability. A. Quantification of TUNEL-positive neurons in response to plasminogen exposure at 36 and 72 h.TUNEL index is expressed as the percentage of TUNEL-positive nuclei to DAPI-stained nuclei (bars represent mean±SD, n=5). *, pb0.0001 versus control. B. DAPI (blue) and TUNEL(red) staining of neurons after long-term incubation (72 h) in the presence or absence of 0.5 µM plasminogen. C. Plasminogen-treated neurons after 72 h of incubation with 0.5 µMplasminogen. Left panel: DAPI staining; middle panel: TUNEL; right panel: active caspase-3 immunofluorescence. The arrow indicates condensed and fragmented nuclei. D. Westernblot analysis of FAK, Fyn and Akt expression in cortical neurons following 72 h incubation with 0.5 µM plasminogen (Pg). Representative photographs of control and plasminogen-treated neurons are shown on top of the panel. In response to Pg treatment, neurons are detached as clusters whereas a homogenous layer of neurons interconnected by a neuriticnetwork is observed for untreated cells. Plasminogen-treated neurons showed reduced amount of FAK, SFK and Akt as compared to untreated-control cells. Bar graphs represent thetotal amount of kinase in plasminogen-treated cells expressed as a percentage+/−sem of total amount of kinase in the control cells set to 100%+/−sem (n=4).

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and rabbit IgGs tomurine tPAor uPAwere obtained and characterized asdescribed (Declerck et al., 1995; Lijnen et al., 1999; Rossignol et al.,2006). Cytarabine (Ara-C), carboxypeptidase B (CpB), anti-tenascin-C mAb, amiloride, trypsin-EDTA and ε-aminocarproic acid, (ε-ACA)were from Sigma (St-Louis, MO). D-Val-Phe-Lys-CH2Cl, 1,5-Dansyl-Glu-Gly-Arg Chloromethyl ketoned HCl (D-GGACK), and N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophanMethylamide (GM6001) were from Calbiochem (La Jolla, CA). Activesite-blocked plasmin was obtained by incubating 10 µM humanplasmin with 2 mM D-Val-L-Phe-L-Lys-CH2Cl at 37 °C for 15min inPBS. Others reagents were obtained as described (Rossignol et al.,2004).

Cell culture

Primary neuronal cultures were established from cerebral corticesobtained from C57BL/6 mice at embryonic day 16. The cells were

seeded in plates coated with 25 µg/mL poly-L-lysine (P9155, Sigma-Aldrich, St-Louis, MO) and cultured at 37 °C in 5% CO2 usingneurobasal medium containing 2% B27 (Invitrogen, USA) and0.5 mML-Glutamine. Half of the media was replaced every 4 days.GFAP-positive astrocytes were less than 2.5% in neuronal cultures andwere eliminated when indicated by supplementing the medium with3 µM Ara-C 24 h after platting the neurons. CHO-K1 cells werecultured at 37 °C in 5% CO2 using Ham's F-12 medium supplementedwith 10% fetal calf serum. CHO-K1 cells were cultured and treatedwith plasminogen as described (Rossignol et al., 2004).

Identification of plasminogen activators and inhibitors

At day 5 after seeding, conditioned medium of mouse corticalneurons was collected and cells were lysed with 1% Triton X-100 inPBS. Conditioned medium and cell lysates were submitted to electro-phoresis under non-reducing conditions in SDS 7.5% polyacrylamide

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gels. Zymography on fibrin-agar gels containing plasminogen andWestern blotting using polyclonal antibodies to mouse tPA wereperformed as described (Rossignol et al., 2004). The tPA activity ofneurons lysates and its inhibition by a blocking antibody tomouse tPAwasmeasured by the fibrin platemethod (Astrup andMullertz, 1952).

Binding and activation of plasminogen onto mouse cortical neurons

Cortical neurons seeded in 96-well plates (105 cells per well) wereincubated with human plasminogen (0 to 5 µM) in culture mediumcontaining 25 µM d-GGACK to prevent plasmin formation. After 2washes with PBS, bound plasminogen was detected with HRP-conjugated mAb CPL-15 specific for plasminogen (Montes et al.,1996). In parallel experiments, neurons were pretreated with varyingconcentrations of carboxypeptidase B to remove COOH-terminallysine residues. Comparative experiments with murine plasminogenwere also performed. Data were fitted according to the Langmuirequation for single site interactions.

For plasminogen activation, cortical neurons seeded as above wereincubated with culture medium supplemented with 0 to 2 µM ofnative plasminogen or of active site mutant recombinant plasminogen(rPg-S741A) and 0.75 mM of CBS0065, a plasmin-selective chromo-genic substrate (Stago, Asnières, France). The inhibitors, ε-ACA (0 to100 mM) or α2-AP (0 to 1 µM) were added to the activation mixtureto investigate their effect on plasmin formation. Ara-C pretreatment ofcultures excluded participation of contaminant astrocytes (less than2.5% GFAP-positive cells) to plasminogen activation by neurons. Toidentify plasminogen activators, cells were pretreated for 1 h with arabbit IgG (20 µg/ml) to murine tPA or uPA or with 200 µM amiloridebefore incubation with 200nM plasminogen and 0.75 mM CBS0065.Kinetics of plasmin formation was followed during 3h as previouslydescribed (Meilhac et al., 2003; Rossignol et al., 2004). In parallelexperiments, cell-conditioned media were analyzed by Westernblotting using HRP-conjugated mAb CPL-15 specific for plasmin(ogen), as indicated (Meilhac et al., 2003).

Effects of plasminogen activation on cell phenotype and survival

The following analyses were performed after the plasminogenactivation experiments. Conditioned medium of cortical neurons wasanalyzed by Western blotting using polyclonal antibodies specific forfibronectin (Biogenesis, UK) or laminin (Abcam, France) and a mAbdirected against tenascin-C (Sigma-Aldrich, St-Louis, MO).

Neuronal clustering and retraction of the cell monolayer in re-sponse to plasmin formation were monitored by contrast phasemicroscopy. Microtiter 96-well plates with plasminogen-treatedand control cells were centrifuged (500 g, 10 min), supernatantswere carefully removed and neuron monolayers were stained with0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT). The MTT-stained neuron microplates were thenscanned and the retraction index was calculated as the ratio of controlto plasminogen-treated MTT-stained neuronal surface area. To quan-titate the extent of detachment, non-adherent cellswere eliminatedbytwo washes in PBS prior to the addition of MTT (0.5 mg/mL) allowingthe detection of residual adherent cells independently of detachedcells. After 1h of incubation at 37 °C, MTT was removed, the formazancrystals dissolved in DMSO and the absorbance measured at 540 nm.

TUNEL (Roche Applied Science, Switzerland) or immunodetectionof active caspase-3 (R&D systems, USA) were performed according tothemanufacturer's instructions on cells dissociated by gentle mechani-cal disruption in the presence of 2.5 mg/ml trypsin and 0.2 mg/mlEDTA, cytospun and fixed in 3.7% paraformaldehyde. The cells werethen counterstainedwithDAPI (Sigma-Aldrich, St-Louis,MO),mountedwith Fluoprep (Dako, Denmark) and observed under an epifluores-cencemicroscope. The TUNEL indexwas calculated as the percentage ofTUNEL-positive nuclei relative to total DAPI-stained nuclei.

Effects of plasminogen activation on signaling pathways

Cells were rinsed twice in cold PBS and quickly frozen on dry ice.Cells were further lysed with boiling SDS (1%) containing NaVO4

(1 mM), immediately sonicated and boiled for 3 min. Proteinconcentration was determined with the BCA assay (Pierce) and 50to 100 µg of protein were analyzed by SDS-PAGE. Immunoblots werecarried out using antibodies specifically directed against the active-phosphorylated form of each kinase or antibodies recognizing eachkinase independently of its state of activation. The followingantibodies were used: anti Phospho-ERK from Sigma-Aldrich(M8159); anti P-Y397-FAK (44-624G) and anti P-Y418-Src (44660-5B) from Biosource (USA); anti ERK (9101), anti P-S473-Akt (4058)and anti Akt (4685) from Cell Signalling Biotechnology (USA); antiFAK from Upstate Biotechnology (USA); anti FYN (sc-16) from Santa-Cruz Biotechnology (USA). Proteins were detected and quantifiedwith the Odyssey Imaging System (LI-COR bioscience, USA). Datawere normalized to the mean value of untreated controls.

Statistical analysis

Results are expressed as means±SD (at least three independentexperiments performed in triplicate unless indicated otherwise).Comparisons used one-way analysis of variance with Scheffe's F test,statistical significance was set at pb0.05. The levels of kinaseactivation were analyzed using unpaired t-test with statisticalsignificance set to: *pb0.05, **0.001bpb0.05, ***pb0.001.

Acknowledgments

We thank Jean-Antoine Girault (Inserm U839) for helpful com-ments on the manuscript. This study was supported by grants fromAssociation pour la Recherche sur le Cancer to HE (ARC-3746) andfrom the Inserm, and the Lower-Normandy Regional Council to EAC.EAC and LD are members of the European Community's SeventhFramework Programme (ARISE grant agreement no. 201024).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi: 10.1016/j.mcn.2009.08.001.

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