浙江大学 文本 Activation of Src tyrosine kinase in microglia in the rat hippocampus following transient forebrain ischemia_生理学

Availeble onfine at wwwsciencedirectcom CiMC图 DIRECT Neuroscience Letters ELSEVTER Neuroseienoe Lemers 380 (2005)1-5 www.clanvizr.comucale/rulat Activation of Src tyrosine kinase in microglia in the rat hippocampus following transient forebrain ischemia Jeong-Sun Choi",Ha-Young Kim,Jin-Woong Chung",Myung-Hoon Chun Seong Yun Kim,Shin-Hee Yoon,Mun-Yong I.ce2.* Drparimw ay Aeuamy:Cullpr Mrieme,The Cahobe Tnneray f gurea.505 Bunp-dng.Surfo-ga Seu 137-701.Konu *myme影a交Cgtg可ledciue.Th Comhate Luer动yE时opoviong Socko-gu Srouf-以.s "Drparew af Pirhoigg.Culryr af Mafein,The Cahot Canrnty af furva 505 Bunpu-dung.Soclu-m Seuaf /37-701.Kora Recerved 6 December 2004:receved n revesed torm 4 mery 2005:accepned 4 January 2005 Abstraet To heller underand the pthopysiulogical mule of Sre proeein.a mon-toxpkor proein lyrssine kineee uf 60kD,in the ischemic hm we imvestignted the rime corse and rrpioral dlistribiiom of nelive Sre:espression hy using a specifie amtibody agains Tyr416 phosphorybalnl Sce (phospho-Sre)in the rat hippocampus afer transient forebrain ischema.In the hippocampus of the control animals,active Sec expression was too low to be detected by immurolabeling Bezmning 4 h after reperfusion,active Sre expression became evident and,after I day.had increned preterentially in the CA feld of the hippocampus proper and the dente gynts By day 3,actie Sre expeession markedly increased in the pyramidal cell lay er of(Al and the demate hilag region in temporal correlation with ne roeal cell death occumng u these areas,where oclls typical uf phapovyti microglia shiwod phoepho-Sre immunureatiity.Double-labcling cxperiments revcald that oclls cxprussing cive Sre were micropias that sincd foe hiutimlald leclin driv from Gr rmp(GSI-B.Aclive Sre xression ben o decline at day 7 and retumed to the basal level by day 14 anter reperfusioe.These results demonstrate increased phophorylatioo of Sre u octivuted microglin ot the poot-ischemi hippocammpus,indicating that Sre sigraling may be inolhved in the microglial reaction to an ischemic nsailt 2004 Fsever Ireland 1id All righs reserved Kiyrontr:Sec;Planpbreylatin Traraiesl fonhrais bclmit,Micili Sre protein,a non-receptor protein tyrasine kinase (PTK) centration and that Sre serves as a signaling protein fixr of 60kDu,is the representative member of the Sre kirase puthological mechanisms of neuronal degenerative disease, family.Cellular Sre(c-Sre),the homolog ofvirus Sre (v-Srel. including ischemia and seizure [11,14].In vitro kinase assarys is widely distributed in marry tissues Dephosphorylation of have shown the ischemia/reperfusicn-induced activution of tyrosine residue 527 (Tyr527)in the C-terminal region may Sre 3].and Ma et al.9]have shown by immunoblotting induce its activation and subeequent autophusphurylation of with antibody against Tyr416 phuspharylated Sre that Tyn416 in its kinaseregion,which further stabilizes its activity Tyr416 phasphorylation of Sre is increased in the rat hip [1.12 pocampus following transient forehrain ischemia.Ilowever. Sre acts as a cummon signaling mediator imolved in a the exact cell phenotype imolved and the passible role of brood speetrum of physological respomses,includine gene active c-Sre an transient forebrain ischemia remaans to be transeriprion,adhesion regulation,and cell differentiation established and survival [1].Recent studies have shown that Sre is To bemer understand the pnthophysiological role of nc nctivnted by signals that inerease intrncellular Ca co- tive Sre i the ischemie brin,we assessed the distribution of active Sre in the rat hippocampus following transient fore- ◆(C0 mespandng1hr1m:4822011球：+8225110 bmin ischemin by using a specife ambody ngainst Tyr416 E-odl aolirp:mury leei callulic.ac k:(M.'t.Lerl phosphorylmed Sre 04-19%0-oe fhort matter 2004 Flarvier Ireland 1d All righes negrred doi:10.1016).neuet.2005.01.01S

Neuroscience Letters 380 (2005) 1–5 Activation of Src tyrosine kinase in microglia in the rat hippocampus following transient forebrain ischemia Jeong-Sun Choia, Ha-Young Kima, Jin-Woong Chunga, Myung-Hoon Chuna, Seong Yun Kimb, Shin-Hee Yoonc, Mun-Yong Leea,∗ a Department of Anatomy, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea b Department of Pharmacology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea c Department of Physiology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea Received 6 December 2004; received in revised form 4 January 2005; accepted 4 January 2005 Abstract To better understand the pathophysiological role of Src protein, a non-receptor protein tyrosine kinase of 60 kDa, in the ischemic brain, we investigated the time course and regional distribution of active Src expression by using a specific antibody against Tyr416 phosphorylated Src (phospho-Src) in the rat hippocampus after transient forebrain ischemia. In the hippocampus of the control animals, active Src expression was too low to be detected by immunolabeling. Beginning 4 h after reperfusion, active Src expression became evident and, after 1 day, had increased preferentially in the CA field of the hippocampus proper and the dentate gyrus. By day 3, active Src expression markedly increased in the pyramidal cell layer of CA1 and the dentate hilar region in temporal correlation with neuronal cell death occurring in these areas, where cells typical of phagocytic microglia showed phospho-Src immunoreactivity. Double-labeling experiments revealed that cells expressing active Src were microglia that stained for biotinylated lectin derived from Griffonia simplicifolia (GSI-B4). Active Src expression began to decline at day 7 and returned to the basal level by day 14 after reperfusion. These results demonstrate increased phosphorylation of Src in activated microglia of the post-ischemic hippocampus, indicating that Src signaling may be involved in the microglial reaction to an ischemic insult. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Src; Phosphorylation; Transient forebrain ischemia; Microglia Src protein, a non-receptor protein tyrosine kinase (PTK) of 60 kDa, is the representative member of the Src kinase family. Cellular Src (c-Src), the homolog of virus Src (v-Src), is widely distributed in many tissues. Dephosphorylation of tyrosine residue 527 (Tyr527) in the C-terminal region may induce its activation and subsequent autophosphorylation of Tyr416 in its kinase region, which further stabilizes its activity [1,12]. Src acts as a common signaling mediator involved in a broad spectrum of physiological responses, including gene transcription, adhesion regulation, and cell differentiation and survival [1]. Recent studies have shown that Src is activated by signals that increase intracellular Ca2+ con- ∗ Corresponding author. Tel.: +82 2 590 1108; fax: +82 2 536 3110. E-mail address: munylee@catholic.ac.kr (M.-Y. Lee). centration and that Src serves as a signaling protein for pathological mechanisms of neuronal degenerative disease, including ischemia and seizure [11,14]. In vitro kinase assays have shown the ischemia/reperfusion-induced activation of Src [3], and Ma et al. [9] have shown by immunoblotting with antibody against Tyr416 phosphorylated Src that Tyr416 phosphorylation of Src is increased in the rat hip￾pocampus following transient forebrain ischemia. However, the exact cell phenotype involved and the possible role of active c-Src in transient forebrain ischemia remains to be established. To better understand the pathophysiological role of ac￾tive Src in the ischemic brain, we assessed the distribution of active Src in the rat hippocampus following transient fore￾brain ischemia by using a specific antibody against Tyr416 phosphorylated Src. 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.01.015

2 J.-S.CNoi e al.Nesrorcieuce LeMers 280 (2095)1-5 Adull male Sprague-Dawley ras (250-300 g)were usod in fiormat,and contrast levels of'images were adjustod using this study.Trarsient forebrain ischemia was induced by four- Adabe Photoshop v.6.0 (Adobe Sysiems Ine..San Jose.CA. vessel occlusion and reperfusion.as previously deseribed by USA Pulsinelli and Brierley [10]with minor modifications [6] Immunohistochemistry demonstrated a low level of Rats were anesthetized with chlral hydrate (400 mg/kg.ip) husal Tyr416 phasphorylated Sre (phospho-Sre)immunore. Briefly,the vertebral arteries were electrocauterized and cut activity in the sham-operated rat hippocampi all time completely to abolish circulation.After 24h.hoth common points (Fig IA and C).Four hours after reperfusion carotid arteries were occluded with miniature aneurism clips (Fig IB),phospho-Sre immunoreactivity was augrnented far 10 min.Only animals with complete electroencephalo in all areas of the hippocampus,and was localized in gram (EEG)flattening upon vascular occlusion were classi cells that exhibited the morpholovical characteristics of fied as ischemic and used in the stuy.Rectal temperature activated microglia (Tig.ID)This expression pattern was maintained at 37.5 10.3"C with a heating lamp during remained uncharged,but the immunoreactivity was further and after ischemin.Sham-opernted rmts with cautenzed ver- enhanced after 1 day (Fig 1E)All immunorenctivity tebenl arteries and lipatures placed around the carotid nrtenes specific to phospho-Sre was climinated when the primary wereused ascontrols Noanimal convulsed or diod following antibody was omitted or substituned with non-specific rabbit repertusion or sham operation.Animals were allowed to live IgG (data not shown).confirming the specificity of this for 4h.12h.or 1.3.7 or 14 days after reperfusion Five mts antibodv. were used for ench time-point Sham-opemted nnimals were At day 3 after reperfusion,phospho-Sre immunoreactiv- treated using the same schedule as the ischemio-reperfused ity wns preferentially increased in the pyramidal cell layer animals.All experimental procedures performed on the ani- of CA1 nnd the dentate hilar region in tempoml correlation mals were conducted with the approval of the Catholic Ethics with neuronal cell death occurring in these areas (Fg.IF). Committee of the Catholic University of Kored.and were in In the pyramidal cell layer of CAl.morphological changes accordance with the Nstioral Institutes of Health Guide for in phospho-Sre immunoreactive cells-loss of cellular pro- the Care and Use of Laboratory Animals (NIH Publication cesses and rounding of the cell body typical of phagocytie N0.80-23.ev1sd1996). microglia-were observed (Fig.IH).At day 7 alter reperfu- At each time point afler reperfusion animals were deeply Bion increased phospho-Sre immunoreactivily was still evi- anesthettzed with 16.9%urethane (10 ml/kgl and killed demt in these regions,but the labeling intensity was decreased by transcardial perfusion with a fixative containing 4% (Fig IG and 1).Al dary 14,active Sre expression had returned paraformaldehyde in D.1 M phosphate bulTer (pH 7.4).Free- 1 o the control level仆ie.IJ) floating sections (25 um thick)were processed for Sre im- Comparison with the distribution of GSI-Ba-positine munuhisstochemistry.After blocking with 10%normal goat cells in the postischemic hippocampus revealed that the serum for I h,the sections were incuhated with a mouse mon- distrihution and the density of phospho-Sre immumoreactive ocloral antibody againe TyT416 phosphorylated Sre (Cal. cells closely correlated with those of activated microglia, biochem,Sa Diego,CA USA;dilued at 1:100)avemight identified by their GSI-B4 labeling (data nat shown)To at 4C.Primary antibody binding wes visualized using confirm the localization of the phospho Sre in activated mi peraxidse-labeled gout ani-mouse antibody (Jacksun Im croglia,double labeled immunofluorescence was performed. munuResearch,West Grove.PA,USA:diluted at 1:200),and Co-labeling with phospho Sre and either GSI-B or von 0.05%3.3'-diaminobengzidine tetrahydrochloride and 0.01% Willehrand factor revealed that phospho-Sre was localized H0 as substrate The specificity of the Sre immunorcactrv- in GSl-Ba-stained microglial cells (Fig 2A-C).burt not in ity wns confined by the absence of'nn immunohistochem- vascular endathelial eells stained for von Willebrnnd factor icnl reaction in sections from which the primary nntibody (Fig 2D-F) ws omitted.or in which it wns substinted with non-specific This is the first demonsrrarion of'the cellular distribution rahhit IgG of phospho-Sre in the rat hippocampus following tmansient For double-immunofluorescence histochemistry.sections forebrnin ischemin Post-ischemic nctivntion of Sre revenled were incubnted in a combination of a mouse monoclonal hy using an nntibody that selectively recognizes tyrosine antibody to Tyr416 phosphorylnted Sre and either a rabhit phosphorylated Sre at its major aophasphorylation site,ty- polyclonal antibody to von Wallebrand factor (Sigma,St rosine 416.was evident within 4 h.reached maximal levels at LouBs.MO.USA.diluted at 1:200)or bictinylated lectin day 3.and was maintained for at least 7 days.Double-labeling (GSI-Ba)derrved from Gnlona simplicifols (Vector Labo- experiments revealed that cells expressing actrve Sre alter rories Inc..CA.USA:diluted at 1:20)ovemightat4C.The ischemie injury were activaled microglia Several in vivo sections were then reacted with Cy3-conjugaled anti-mouse studies have recently repored ischemis'reperfusson-induced antbody (Jackson.diluted at1:500)o FTTC-corpugated ant actraton of Sre in the rat hippocampus by immunoblolling botin Ig (Jackson,diluted at 1:50)respectively.for 2h 19]and in vitro kinase assay 13J.Thus,our data remforce the at room temperature.Control sections were prepared as de- idea that Sre is activaled in the ischemie brain.and provide in seribed above.Sldes were vewed using a confocal micro- situ localzation of tyrosine phosphorylated Sre in actrvated scope (MRC-1024,BioRad).Images were comverted to TIFF microglia

2 J.-S. Choi et al. / Neuroscience Letters 380 (2005) 1–5 Adult male Sprague-Dawley rats (250–300 g) were used in this study. Transient forebrain ischemia was induced by four￾vessel occlusion and reperfusion, as previously described by Pulsinelli and Brierley [10] with minor modifications [6]. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.). Briefly, the vertebral arteries were electrocauterized and cut completely to abolish circulation. After 24 h, both common carotid arteries were occluded with miniature aneurism clips for 10 min. Only animals with complete electroencephalo￾gram (EEG) flattening upon vascular occlusion were classi- fied as ischemic and used in the study. Rectal temperature was maintained at 37.5 ± 0.3 ◦C with a heating lamp during and after ischemia. Sham-operated rats with cauterized ver￾tebral arteries and ligatures placed around the carotid arteries were used as controls. No animal convulsed or died following reperfusion or sham operation. Animals were allowed to live for 4 h, 12 h, or 1, 3, 7 or 14 days after reperfusion. Five rats were used for each time-point. Sham-operated animals were treated using the same schedule as the ischemic-reperfused animals. All experimental procedures performed on the ani￾mals were conducted with the approval of the Catholic Ethics Committee of the Catholic University of Korea, and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80–23, revised 1996). At each time point after reperfusion, animals were deeply anesthetized with 16.9% urethane (10 ml/kg) and killed by transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Free- floating sections (25
m thick) were processed for Src im￾munohistochemistry. After blocking with 10% normal goat serum for 1 h, the sections were incubated with a mouse mon￾oclonal antibody against Tyr416 phosphorylated Src (Cal￾biochem, San Diego, CA, USA; diluted at 1:100) overnight at 4 ◦C. Primary antibody binding was visualized using peroxidase-labeled goat anti-mouse antibody (Jackson Im￾munoResearch, West Grove, PA, USA; diluted at 1:200), and 0.05% 3,3 -diaminobenzidine tetrahydrochloride and 0.01% H2O2 as substrate. The specificity of the Src immunoreactiv￾ity was confirmed by the absence of an immunohistochem￾ical reaction in sections from which the primary antibody was omitted, or in which it was substituted with non-specific rabbit IgG. For double-immunofluorescence histochemistry, sections were incubated in a combination of a mouse monoclonal antibody to Tyr416 phosphorylated Src and either a rabbit polyclonal antibody to von Willebrand factor (Sigma, St. Louis, MO, USA; diluted at 1:200) or biotinylated lectin (GSI-B4) derived from Griffonia simplicifolia (Vector Labo￾ratories Inc., CA, USA; diluted at 1:20) overnight at 4 ◦C. The sections were then reacted with Cy3-conjugated anti-mouse antibody (Jackson; diluted at 1:500) or FITC-conjugated anti￾biotin IgG (Jackson; diluted at 1:50), respectively, for 2 h at room temperature. Control sections were prepared as de￾scribed above. Slides were viewed using a confocal micro￾scope (MRC-1024, BioRad). Images were converted to TIFF format, and contrast levels of images were adjusted using Adobe Photoshop v. 6.0 (Adobe Systems Inc., San Jose, CA, USA). Immunohistochemistry demonstrated a low level of basal Tyr416 phosphorylated Src (phospho-Src) immunore￾activity in the sham-operated rat hippocampi at all time points (Fig. 1A and C). Four hours after reperfusion (Fig. 1B), phospho-Src immunoreactivity was augmented in all areas of the hippocampus, and was localized in cells that exhibited the morphological characteristics of activated microglia (Fig. 1D). This expression pattern remained unchanged, but the immunoreactivity was further enhanced after 1 day (Fig. 1E). All immunoreactivity specific to phospho-Src was eliminated when the primary antibody was omitted or substituted with non-specific rabbit IgG (data not shown), confirming the specificity of this antibody. At day 3 after reperfusion, phospho-Src immunoreactiv￾ity was preferentially increased in the pyramidal cell layer of CA1 and the dentate hilar region in temporal correlation with neuronal cell death occurring in these areas (Fig. 1F). In the pyramidal cell layer of CA1, morphological changes in phospho-Src immunoreactive cells—loss of cellular pro￾cesses and rounding of the cell body typical of phagocytic microglia—were observed (Fig. 1H). At day 7 after reperfu￾sion, increased phospho-Src immunoreactivity was still evi￾dent in these regions, but the labeling intensity was decreased (Fig. 1G and I). At day 14, active Src expression had returned to the control level (Fig. 1J). Comparison with the distribution of GSI-B4-positive cells in the postischemic hippocampus revealed that the distribution and the density of phospho-Src immunoreactive cells closely correlated with those of activated microglia, identified by their GSI-B4 labeling (data not shown). To confirm the localization of the phospho-Src in activated mi￾croglia, double-labeled immunofluorescence was performed. Co-labeling with phospho-Src and either GSI-B4 or von Willebrand factor revealed that phospho-Src was localized in GSI-B4-stained microglial cells (Fig. 2A–C), but not in vascular endothelial cells stained for von Willebrand factor (Fig. 2D–F). This is the first demonstration of the cellular distribution of phospho-Src in the rat hippocampus following transient forebrain ischemia. Post-ischemic activation of Src, revealed by using an antibody that selectively recognizes tyrosine phosphorylated Src at its major autophosphorylation site, ty￾rosine 416, was evident within 4 h, reached maximal levels at day 3, and was maintained for at least 7 days. Double-labeling experiments revealed that cells expressing active Src after ischemic injury were activated microglia. Several in vivo studies have recently reported ischemia/reperfusion-induced activation of Src in the rat hippocampus by immunoblotting [9] and in vitro kinase assay [3]. Thus, our data reinforce the idea that Src is activated in the ischemic brain, and provide in situ localization of tyrosine phosphorylated Src in activated microglia

L-5 Choi er al.NVeuroschence Leters 390 (2505)1-5 Fig.1.Chnses in phoopho-Sie immaraea:tvay in the rat hippocampes following trrtsicn fiedbrain icherin.In the cobol woclun (Al,no sianficart phossho-Sre mmnanoreactwviy was observed in the rat hippocampus.Ater 4hot reperfision (3).phospho-Sr mmunoreactrm wasevident in the dendrnc layer.o,the critem rrimns g.the dranin radiatam Afer I dry o rperfurn ()phoepho-Sr irnereartnity Fad incre.vnd nather homngenoasly over the hippecamun.Afer days of repefinion (Fl.phoaph-immiiy had incieaad prefcertialy n CAl and the dertate hlar ni.Ry thry 7 1 the expresson of phospho-sh3对decreased and had returned1 the control level by day14八(Ha对I》Higher m2ta边os6 the bosed are2 s thom F un!G.topectivch.Sdle leas-300pm fot A,B.E.F and Gi,100 um fu C,D,H anL

J.-S. Choi et al. / Neuroscience Letters 380 (2005) 1–5 3 Fig. 1. Changes in phospho-Src immunoreactivity in the rat hippocampus following transient forebrain ischemia. In the control section (A), no significant phospho-Src immunoreactivity was observed in the rat hippocampus. After 4 h of reperfusion (B), phospho-Src immunoreactivity was evident in the dendritic layers of the hippocampus proper and the dentate gyrus. (C and D) Higher magnifications of the boxed areas from A and B, respectively. pcl, the pyramidal cell layer; so, the stratum oriens; sr, the stratum radiatum. After 1 day of reperfusion (E), phospho-Src immunoreactivity had increased rather homogenously over the hippocampus. After 3 days of reperfusion (F), phospho-Src immunoreactivity had increased preferentially in CA1 and the dentate hilar region. By day 7 (G), the expression of phospho-Src had decreased and had returned to the control level by day 14 (J). (H and I) Higher magnifications of the boxed areas from F and G, respectively. Scale bars = 300
m for A, B, E, F and G; 100
m for C, D, H and I

d-星Chu e星/reschenee Lelle50a99J- B (E)in gieen (C.FI Supenrposed mazes of Cy3 (A,D)and FITC [B.E)Note thet phospo-Sre ummanoceactivgy wes oolocelized in microglia stained for G51-Ba.bt m in vancalar endrtheal cells(amw)xained for von Wilchtand faclor.pel,the pytarridd cell laye,the sntun nitun.Seale hrs50pr for A- Active Sre expression wis accomparted by morpbolegi- an interaction with PYK2.may be involved in mcroglial cal changes in the microglia By day I after ischemic injury activution in the ischemic hippocampus.Honwever,the phospho-Sre immunoreactive cells had the morphology of importance of the signaling cascades imvolving Sre in activated microglia,ie,contraction of their highly ramified reactive microglia has yet to be clarified. processes,and therehy appeured souter in shape At day 3. In cooclusion,we hae demonstruted that phosphoryla- active Sre expression was also observed in phagocytic mi- tion of Sre is markedly up regulated in activated microglia in croglia in the pyramidal cell layer of the CAl region,where the rat hippocampus after transient forebrain ischemia.These severe neuronal death occurred because of the ischemic in. results suggest that Sre signaling is involved in microglial ac- jury.These results are consistent with the obsenvation that tivation in response to ischemic injury. an early generalized miroglial reaction in all hippocampal subficlds was followed by a degeneration-specific reaction in the areas with neural degeneration after ischemic ingury 141 Acknuwledgment Recently.enhaneed imeracton of'Sre and proline-nch tyrosine kinase 2 (PYK2)another non-receptor tyrosine This research was supported by a grant (M103KV0103 kirase.with N-methyl-D-aspartate (NMDA)receptor 1903K22010193)from Brain Rescarch Center of the 21st subunit 2A (NR2A]has been shown to be induced by Century Fromtier Research Program furded by the Ministry ischemin/reperfusion and promote the influr of Caz of Science and Technology of Republic of Koren through the NMDA receptor and up regulation of NMDA receptor function [7-9].It has been reported that tyrosine phosphorylated PYK2 was observed in micruglia in resporse References to CNS insults 12.131 Tian et al [13]demanstrated the pronounced and sustained phosphorylstion of PYK2 in [川M.T.eu黑，.A.Cooe,Regalation,substrales and fanxtons of c,Biuhin.Buplrys Aciu 287 (1905)121-149. microglial cells after focal cerebral iscbemia and seizure, suggesting thaut PYK2 is imolved in microglial activation C.K.Combs.DE Jhson,SB.Canaty.T'M.Lchmn,Gll Lan ae小Ideti山e0 of mirogdial sigal uurductin petlweys me. as an upstream regulalor of p3s milogen-activsled protein dating a nomtosi:tore to artylnidngmi:fragmets of beta- kinase (MAPK)Oxidsnt stress gereraled during global amyfoid and pnon protzins,J.Neurosci.19 (1599)928-939. ischemiareperfuson activaled p38 MAPK in macrophage [3]I Giu,F.Meng.G Zleng Q.Zhang.Fiee nlials ae inubel m comtiruoss astivanon of ronreceptor tyrogne pronein kirase c-Sre cell lines through a Sre-dependent pathway [5]Considered aner ishemiafreperfsin in hispomp,Nounoci.e 345 together,our data suggest that Sre activation,possibly by 10均101-104

4 J.-S. Choi et al. / Neuroscience Letters 380 (2005) 1–5 Fig. 2. Identification of phospho-Src in the hippocampus 3 days after forebrain ischemia. Phospho-Src (A, D) was in red, GSI-B4 (B) and von Willebrand factor (E) in green. (C, F) Superimposed images of Cy3 (A, D) and FITC (B, E). Note that phospho-Src immunoreactivity was colocalized in microglia stained for GSI-B4, but not in vascular endothelial cells (arrows) stained for von Willebrand factor. pcl, the pyramidal cell layer; sr, the stratum radiatum. Scale bars = 50
m for A–F. Active Src expression was accompanied by morphologi￾cal changes in the microglia. By day 1 after ischemic injury, phospho-Src immunoreactive cells had the morphology of activated microglia, i.e., contraction of their highly ramified processes, and thereby appeared stouter in shape. At day 3, active Src expression was also observed in phagocytic mi￾croglia in the pyramidal cell layer of the CA1 region, where severe neuronal death occurred because of the ischemic in￾jury. These results are consistent with the observation that an early generalized microglial reaction in all hippocampal subfields was followed by a degeneration-specific reaction in the areas with neural degeneration after ischemic injury [4]. Recently, enhanced interaction of Src and proline-rich tyrosine kinase 2 (PYK2), another non-receptor tyrosine kinase, with N-methyl-d-aspartate (NMDA) receptor subunit 2A (NR2A) has been shown to be induced by ischemia/reperfusion and promote the influx of Ca2+ through the NMDA receptor and up regulation of NMDA receptor function [7–9]. It has been reported that tyrosine￾phosphorylated PYK2 was observed in microglia in response to CNS insults [2,13]. Tian et al. [13] demonstrated the pronounced and sustained phosphorylation of PYK2 in microglial cells after focal cerebral ischemia and seizure, suggesting that PYK2 is involved in microglial activation as an upstream regulator of p38 mitogen-activated protein kinase (MAPK). Oxidant stress generated during global ischemia/reperfusion activated p38 MAPK in macrophage cell lines through a Src-dependent pathway [5]. Considered together, our data suggest that Src activation, possibly by an interaction with PYK2, may be involved in microglial activation in the ischemic hippocampus. However, the importance of the signaling cascades involving Src in reactive microglia has yet to be clarified. In conclusion, we have demonstrated that phosphoryla￾tion of Src is markedly up regulated in activated microglia in the rat hippocampus after transient forebrain ischemia. These results suggest that Src signaling is involved in microglial ac￾tivation in response to ischemic injury. Acknowledgment This research was supported by a grant (M103KV0100 1903K220101930) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Republic of Korea. References [1] M.T. Brown, J.A. Cooper, Regulation, substrates and functions of src, Biochim. Biophys. Acta 1287 (1996) 121–149. [2] C.K. Combs, D.E. Johnson, S.B. Cannady, T.M. Lehman, G.E. Lan￾dreth, Identification of microglial signal transduction pathways me￾diating a neurotoxic response to amyloidogenic fragments of beta￾amyloid and prion proteins, J. Neurosci. 19 (1999) 928–939. [3] J. Guo, F. Meng, G. Zhang, Q. Zhang, Free radicals are involved in continuous activation of nonreceptor tyrosine protein kinase c-Src after ischemia/reperfusion in rat hippocampus, Neurosci. Lett. 345 (2003) 101–104

and kain s,Exp of p38 n 161 M.-Y Lee.SLShin.Ys.Choi.EJ Km.H Cha.M-H.Chur S.B.Lee,S.Y.Kim,Tran 26w 2

J.-S. Choi et al. / Neuroscience Letters 380 (2005) 1–5 5 [4] M.B. Jorgensen, B.R. Finsen, M.B. Jensen, B. Castellano, N.H. Diemer, J. Zimmer, Microglial and astroglial reactions to ischemic and kainic acid-induced lesions of the adult rat hippocampus, Exp. Neurol. 120 (1993) 70–88. [5] R.G. Khadaroo, J. Parodo, K.A. Powers, G. Papia, J.C. Marshall, A. Kapus, O.D. Rotstein, Oxidant-induced priming of the macrophage involves activation of p38 mitogen-activated protein kinase through an Src-dependent pathway, Surgery 134 (2003) 242–246. [6] M.-Y. Lee, S.-L. Shin, Y.-S. Choi, E.J. Kim, J.-H. Cha, M.-H. Chun, S.B. Lee, S.Y. Kim, Transient upregulation of osteopontin mRNA in hippocampus and striatum following global forebrain ischemia in rats, Neurosci. Lett. 271 (1999) 81–84. [7] Y. Liu, X.Y. Hou, G.Y. Zhang, T.L. Xu, L-type voltage-gated calcium channel attends regulation of tyrosine phosphorylation of NMDA receptor subunit 2A induced by transient brain ischemia, Brain Res. 972 (2003) 142–148. [8] Y. Liu, G. Zhang, C. Gao, X. Hou, NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A(NR2A) and interaction of Pyk2 and Src with NR2A after transient cerebral ischemia and reperfusion, Brain Res. 909 (2001) 51–58. [9] J. Ma, G.Y. Zhang, Y. Liu, J.Z. Yan, Z.B. Hao, Lithium suppressed Tyr-402 phosphorylation of proline-rich tyrosine kinase (Pyk2) and interactions of Pyk2 and PSD-95 with NR2A in rat hippocam￾pus following cerebral ischemia, Neurosci. Res. 49 (2004) 357– 362. [10] W.A. Pulsinelli, J.B. Brierley, A new model of bilateral hemi￾spheric ischemia in the unanesthetized rat, Stroke 10 (1979) 267– 272. [11] Y. Seko, K. Tobe, N. Takahashi, Y. Kaburagi, T. Kadowaki, Y. Yazaki, Hypoxia and hypoxia/reoxygenation activate Src family ty￾rosine kinases and p21ras in cultured rat cardiac myocytes, Biochem. Biophys. Res. Commun. 226 (1996) 530–535. [12] F. Sicheri, J. Kuriyan, Structures of Src-family tyrosine kinases, Curr. Opin. Struct. Biol. 7 (1997) 777–785. [13] D. Tian, V. Litvak, S. Lev, Cerebral ischemia and seizures induce tyrosine phosphorylation of PYK2 in neurons and microglial cells, J. Neurosci. 20 (2000) 6478–6487. [14] X.M. Yu, M.W. Salter, Src, a molecular switch governing gain con￾trol of synaptic transmission mediated by N-methyl-D-aspartate re￾ceptors, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7697–7704