Neuroscience 123 (20041 147-154 9 NEUROPROTECTIVE ROLE OF EXTRACELLULAR SIGNAL- REGULATED KINASE IN N-ACETYL-O-METHYLDOPAMINE-TREATED HIPPOCAMPAL NEURONS AFTER EXPOSURE TO IN VITRO AND IN VIVO ISCHEMIA E.M.PARK,T.H.JOH,B.T.VOLPE,C.K.CHU, K可 ward: ERK and CREB phaxphorylation, G.SONG*ANDS.CHO■ neuroprotection.Nacetyl-O-methldopamine,sekctive neu "Depordment of Neurology ond Nesroscience,Wel Medloal Colepe of ronal injury,oxygen-glucose deprivation,transient forebrain ischemia. Pit丝，Y1CeC5USA Upon pathological strees,cels activale multiple signaling x力y行maG.AnK行A1002l5A patmays that delemmine their fate.Although the inhibition of targeted death cascadas oould provide neuraprotection, promoong the endogenous survivalrepair mechaniam may xurvivalirepair psthwxys in addition to dexth caxcadex.Acti bear equal,if not greater.importance in achieving neuro vation of cyclk AMP-response-ckment-binding protein (CREB)is linked to nesroprotection in experimental animal Achivation of cydlic AMP-resporse-alement binding models of stroke.However,a role of the mitogen-activated protein kinasefextracelular signal-rogulated kinase kinase protein (CREB)by phosphorylstion at the Ser133 residue (MAPKIERK or MEK),an upatrsam kinass for CREB,and itx is crtical in regulating the axpression of many downstream ralation to CREB phasphorylation in nauroprataction in cn- genes containing CRE elements (Frank and Greenberg. rebral ischemia has not been delneated.Previously,we re 1994:Davis nt al.2000).CREB is phosphorylated by ported that N-acety-O-methyldopamine (NAMDA]signii mutiple kinases including prolein kinase A.cakium-ca candy provected CA1 neurons after transient forebrain isch modulin kinases.prolein kinase B (PKB)(Du and Mor emia [J Neurosci 19 (1999b)87.8]The current study is to miny,1996)and mitogen-actvated proben kinasefextracel imvastigata whethar NANOA-inducad neurcprobaclion occur via the aetivation of ERK and its downstream affeetor,CREB. lular signal regulated kinase kinase (MAPK/ERK.MEK)via NAMDA induced ERK112 and CRE phosphorylation with nibosomal S6 kinase(RSK).Functicnally,CREB phosphor incrasead survival of HC252 hippocampal naurong subjactnd ylation is involved in nerve cell excitation,CNS develop- to oxygen-glucose deprivation.These effeats were reversed ment,long-tem memcry fomation,and circadian rhythm by UD126.a MEK kinase inhibitor.Similary.animals treated as wel as neuroprotecton [Waton et al.,1998,1999 with NAMDA following ischemia showed increased ERK and Ricno at Al.,1999 Somars at al 1995:Tanaka et al. CREB phosphoryfation in the CA1 subregion of the hip- pooampus during carty reperfusion period with increased 1999,2000:Wallon and Dragunow,2000:Mabuchi et al., numher of survhring raurons examined days following 2001)In vittm studes suggesind that CREB regulates ischemia.The NAMDA-induced rauroprotection wid abol genes that mediale neurotrophin-induced neuronal sur- ished by U9126 administered shorthy atter repertusion.The vival (Bonni et aL.1999:Riocio et al.,1999).In an animal esults ghowed that the ERK-CREB 8妇nngn时hT海mgm model of transient forebrain ischemia,where CA1 hip be inwolyod in NAMDA-nduced ncuroprotoction following pocampal neurons degenerate selectively (Kirino,1982: transient global ischemia and imply that the activation of the Pulsneli,1985),the degree of CREB phosphorylatian to treat stroke or other neurological syndromes.2003 (pCREB)w8s positiely comelaned with surviing neurons IBRO.Published by Eisavier Lhd.All rights recerved and alo shoen to be 8 protective response (Walton et al.. 'Comrspendine e:8.Che,Well Mecical Colleps of Cemal Univr. 1998:Hu et aL,1999;Mabuchi et al.,2001).Moreower.a aty,NeurclogyNouioeiology.411 Eit e2th Sreet New York NY neumprotectiva mle of pCREB has bean racantly dafinad 101,AT+1-212-570-3t6e:+1-312--3872 by disrupting of CREB function in the mouse CNS (Man- E.mal address:5uc2002med comeledu [S.Chol. tamadiotis nt al.,2002) AhbredaNons:ANCVA ansheis af vartarce:bFGF hasic thmblaet clemenl-binding protein MEK,an upstrnam kinase of CREB,is a momber of the DB.3.3'daminobenzidine:DMEM.Dubecco's Modifed Eagle MAPK family that is involved in cel cimerentiation and meduum;DS0,dimnetlrdl suphacide,ERx1/2,enloolulir signd- prolferabon,learning.long-temm pateneat on,and neuronal 1/2:MAPKERK cr MEK mingen-aetstns k nasslexirceluisr好tkrarilrled kirscon付rou NANGA plesticity (English and Sweat 1996.1997:Alkins et al.. ,3次+-To7行00mn：.C0门2型Ce-t:■5DG 1998:Impey ctal1999:Darvis et al.,2000).Unlke CREB, 比e role of the MEK pathway in pathologic insuls su边g saline:pCREB,phosphorylaled CREB:pERK phosphorylaled ERK. carehral ischemia is controvergial.Whereas the inhibition PLSD,prolicbd lisl signfcrt dfernot;RSK,rbesami 8a 8D8.sodium dodecyl;TB3,tia-buferod of MEK pathwary was shown to be either neuroprotective or fnur-en nechitinn hed no affect in ischem a (Alassandrini at al.,1559;Sugino C308-452210459000-0c0 2009 ERC.Puetaed by Eaevor Lid.Al niglrs nsorvod. dct10.1016e5en802030g023 147
A NEUROPROTECTIVE ROLE OF EXTRACELLULAR SIGNALREGULATED KINASE IN N-ACETYL-O-METHYLDOPAMINE-TREATED HIPPOCAMPAL NEURONS AFTER EXPOSURE TO IN VITRO AND IN VIVO ISCHEMIA E. M. PARK,a T. H. JOH,a B. T. VOLPE,a C. K. CHU,b G. SONGb AND S. CHOa * a Department of Neurology and Neuroscience, Weill Medical College of Cornell University College at W. M. Burke Medical Research Institute, White Plains, NY 10605, USA b Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, GA 30602, USA Abstract—In response to cerebral ischemia, neurons activate survival/repair pathways in addition to death cascades. Activation of cyclic AMP-response-element-binding protein (CREB) is linked to neuroprotection in experimental animal models of stroke. However, a role of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK/ERK or MEK), an upstream kinase for CREB, and its relation to CREB phosphorylation in neuroprotection in cerebral ischemia has not been delineated. Previously, we reported that N-acetyl-O-methyldopamine (NAMDA) signifi- cantly protected CA1 neurons after transient forebrain ischemia [J Neurosci 19 (1999b) 87.8]. The current study is to investigate whether NAMDA-induced neuroprotection occurs via the activation of ERK and its downstream effector, CREB. NAMDA induced ERK1/2 and CREB phosphorylation with increased survival of HC2S2 hippocampal neurons subjected to oxygen-glucose deprivation. These effects were reversed by U0126, a MEK kinase inhibitor. Similarly, animals treated with NAMDA following ischemia showed increased ERK and CREB phosphorylation in the CA1 subregion of the hippocampus during early reperfusion period with increased number of surviving neurons examined 7 days following ischemia. The NAMDA-induced neuroprotection was abolished by U0126 administered shortly after reperfusion. The results showed that the ERK-CREB signaling pathway might be involved in NAMDA-induced neuroprotection following transient global ischemia and imply that the activation of the pathway in neurons may be an effective therapeutic strategy to treat stroke or other neurological syndromes. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ERK and CREB phosphorylation, neuroprotection, N-acetyl-O-methyldopamine, selective neuronal injury, oxygen-glucose deprivation, transient forebrain ischemia. Upon pathological stress, cells activate multiple signaling pathways that determine their fate. Although the inhibition of targeted death cascades could provide neuroprotection, promoting the endogenous survival/repair mechanism may bear equal, if not greater, importance in achieving neuroprotection. Activation of cyclic AMP-response-element binding protein (CREB) by phosphorylation at the Ser133 residue is critical in regulating the expression of many downstream genes containing CRE elements (Frank and Greenberg, 1994; Davis et al., 2000). CREB is phosphorylated by multiple kinases including protein kinase A, calcium-calmodulin kinases, protein kinase B (PKB) (Du and Montminy, 1998) and mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MAPK/ERK, MEK) via ribosomal S6 kinase (RSK). Functionally, CREB phosphorylation is involved in nerve cell excitation, CNS development, long-term memory formation, and circadian rhythm as well as neuroprotection (Walton et al., 1996, 1999; Riccio et al., 1999; Somers et al., 1999; Tanaka et al., 1999, 2000; Walton and Dragunow, 2000; Mabuchi et al., 2001). In vitro studies suggested that CREB regulates genes that mediate neurotrophin-induced neuronal survival (Bonni et al., 1999; Riccio et al., 1999). In an animal model of transient forebrain ischemia, where CA1 hippocampal neurons degenerate selectively (Kirino, 1982; Pulsinelli, 1985), the degree of CREB phosphorylation (pCREB) was positively correlated with surviving neurons and also shown to be a protective response (Walton et al., 1996; Hu et al., 1999; Mabuchi et al., 2001). Moreover, a neuroprotective role of pCREB has been recently defined by disrupting of CREB function in the mouse CNS (Mantamadiotis et al., 2002). MEK, an upstream kinase of CREB, is a member of the MAPK family that is involved in cell differentiation and proliferation, learning, long-term potentiation, and neuronal plasticity (English and Sweat 1996, 1997; Atkins et al., 1998; Impey et al., 1999; Davis et al., 2000). Unlike CREB, the role of the MEK pathway in pathologic insults such as cerebral ischemia is controversial. Whereas the inhibition of MEK pathway was shown to be either neuroprotective or had no effect in ischemia (Alessandrini et al., 1999; Sugino *Correspondence to: S. Cho, Weill Medical College of Cornell University, Neurology/Neurobiology, 411 East 69th Street, New York, NY 10021, USA. Tel: 1-212-570-2900; fax: 1-212-988-3672. E-mail address: suc2002@med.cornell.edu (S. Cho). Abbreviations: ANOVA, analysis of variance; bFGF, basic fibroblast growth factor; CREB, cyclic AMP-response-element-binding protein; DAB, 3,3-diaminobenzidine; DMEM, Dulbecco’s Modified Eagle medium; DMSO, dimethyl sulphoxide; ERK1/2, extracellular signalregulated kinase 1/2; MAPK/ERK or MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NAMDA, N-acetyl-O-methyldopamine; OD, optical density; OGD, oxygen-glucose deprivation; PB, phosphate buffer; PBS, phosphate-buffered saline; pCREB, phosphorylated CREB; pERK, phosphorylated ERK; PLSD, protected least significant difference; RSK, ribosomal S6 kinase; SDS, sodium dodecyl sulphate; TBS, tris-buffered saline; 4-VO, four-vessel occlusion. Neuroscience 123 (2004) 147–154 0306-4522/04$30.000.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.08.023 147
1-48 E.M Park al.Neresciene 123 (2001)147-154 at al.,2000 Namura et al..20011 MEK acthvation was examine neuronal morphology.HC252 neurons (1x 10]in cham inked to neuroprotection in ischemic tolerance and hypo- ber sldes afer 12 h OGD wore fixed wth 2%col fommaldehyde thermia.and in estrogen freatment against ecitotoic le- in 0.1 M sodium phosphate bufer (FB:pH 7.2),and stained wih sions (Hicks et al.,2000;Strohm et al.,2000;Kuraki et al., 01.1%Creoyl Viniel 20011. We previously reported that treating animals with N Wastom blot hybridization acetyl-C-methyldopamine (NAMDA)increased the number Cwg1×i内ww时washed with phoes-igw时gr附P of surviving CA1 neurcns after transient global ischemis 0.1 M pH 7.41,colkcoed,and ocntritugod to pelet To lyse the ocl (Cho et al..1998b).The purpas8 of the curant shudy is to pelkt 0.5 ml of RIPA buffer (1x FBS,1%NP40.0.5%socium astablish a role for ERK and ts downstraam CREB signal deoxycholata,01%sedium dodncy sulphate (50S]wih freshly ing in neuroprolection folowing ischemia.Specifically.we added shemymethyl suphonyl fuoride PMSF 0.4 mV)aprotnin focused to invnstigate whnther NAMDA-induced nouropro- 0ulm单nd对um urhowaradse(1与were adad3rhw pollt,incubated for 30 min on ke,cenorfuged at 1000xg for 16 inction ocours via an ERK-CREB patmway in vro (oo min at 4 "C.Prooein ooncentradion from the supernstant was ygen glucose deprivation.OGD)and in vivo ischemic con- detamined (Bio-Rad Laberatorias.Herculs,CA.LSA)and 30 ug drtions. of prole ns were losded for SLS-polyacryismide gel electophore- is (PAGE)Electrophorusis was perlormed and prolans were EXPERIMENTAL PROCEDURES transforred from the gel to low protein binding Durapore (PVDF Matarials an electrobloming apparatus.Memhranes were blocked ovemight n1s-butered网n8TB的n侧nmn30.1%1wee20ad5% Coll oulture media and andbictics wore obtaincd from Media Tech (Horndon,VA,USA)N2 supplcment basio fibroblast growth foo- (1:10001,pERK(1:2000)and c-Fos(1:1000:Oncopene Rescarch er [hFGF)Bartamcin,rd furcinn war purchnd frem Produd我.Cambridga MA.l3A.包ownd by horaRt适hpw Gboo (Grand biland,NY.USA)Poly4-omthine and laminin wer idase-conjugaled secondary ansbocie for 1 h.Membranes were obtained from Sigma (St.Louis,MO.USA)and BD Bicscionce ah5deeT对830mne寸7whT85Gu3n301% (Ecdford,MA.USA)respccdvely.NAMDA was syrthesizod from Twccn-20 betwcen coch step.Procein bands were vsuaized by methyldopamine hdrochorice (Aldrich Chcmical Co.,Miwaukee, an ECL Plus Westem bloting devcodon system (Amersham Fhar Wl,LSA)areerding In the mathed dascrbed pravinualy by Cha at mca Binlech e.).The mambrana ix Ablatad In viuia non- al.(1909b).The antbodias drecoad against phesphoryanad phosphoryaad form of ERK (1:1000)and CREB (1:1000 Fo Sert33 and phosphoryation stanc-indaperdent CREB were ob- quantficaton,denstes of phosphorysled ERK and CHEB were taincd from Upstate Elotchnology (Lake Flacid,NY,USA).The normalzed by corespording non-phosphorylaned foms of ERK and CREB.The values were then expressed in rclaton to that of and phasphorylation stale-indepandent ERK anbodi wira pur- non-OGD chasod from Santa Cruz Biotochnology (Sama Cnuz,CA.USAl. UO125 (a MEK1/2 inhibitork SB203580 (a p38 MAFK inhibitorl. Transient forebrain ischemia LY294002 (a phoophatidylinoshol 3(P13)kinase inhibtor)and Animal sirgary was in enm年anca with tha Nain作Inslnae of Healt Cuida fer the Care and Lan cf Laberanery Animals and The Cell eultures Instiutional Animel Care and Use Commitlee ol Weil Medical Collge of Comel University.Al efforts were made to minimize The mmortalzed neuronal progenior cel lne HC252 was derved the number of animals used and ther s.ffering.Aduit male Wistar fom adut rat hippocampus (Hoshimaru et a.,1990)and was mw200-250年：Hil Tep.S1nn,AZ山BAl were A-Wn- obta n时元mCr.Fred G8e闭h83 8k Ingtul(San,CA 1位d wi a miure or halo管anm(1%.ax9的and ritog9nard 8.HC2S2G6gw网grown in gen-rw8Du由gkf时 u四aly prepare6d句r four-vessel ooc80n6-8o8dngo Eagle medium (DMEMUHam's /Ham's F.12 (5050)with glu the mcthod described by Pusincli ct al.(1982 Erafy,both temine contaiing N2 supplment (nsulin at 5 ugml,human tranislern at 50 ugl.20 nM progesterone.100 uM pufrescine. common carotd arries wore cocluded using revers bl dasps O rM godium gelenile).tGF a1 2 ngml.gentsmica and fung lateral blood faw to the brain was controled by an odjustsble neck and laminin.Cels were splt when fey were 70-80%confuerL suture.A burrhole on the skull(1 mm poeslenor to bregma.1.5 mm lateral)was drlied for post-ischemie administration of U0125 ar OGD wchide after ischemia.Animal were deprved of food overnigh hut waler was fraal avsilbl On ta f:llowing day,10 min 4-VD This kylam的iloE nega an bchemi:ke fa:ndlion in wimm cheTa nduced by女htening the c5 a'ound them Culture HC252 oall (1x10)Rra Faplaced with sither gucnsa Tm3ncg时anes3ndhe8丸ue.To mnimized varisb偏，.te comaining or glucose-free DMEM/Ham's F-12 (50/50)and treanad following orteria were applicd:loss of righting refex and bilater with ciher one or combination of folowing drugs:NAMDA5mM) U012520l,9E2035g010o1Y234002100.Thc poithamic coma Aar 10 min bthetia Anmals wara Iragled! cuturas wera mmadialaly pla4dinA中atnf《Ele4-Ra中hn wth either triple in ection of saline or NAMDAA(10 mgkgl at O.1/2. berg.Dal Mar,CA.USA).Except non-03D controla,chambers conaining cubures for O3D were fushed with 95%N 5%CO. Echemio coma,5 pl or 100 pM L0125 (total 0.5 nmoles)or wchick oas mboourc tor 15 min at a ow rate of 4 I per minute (LPMJ to 04高dmohy阳phos.DC的wers injecled over5min城rs create a condton.Both normoodc and chambers laral vartri时mS5 m Imm dira)The body lam年PArRre of all were incbaed at an 37 'C inciharer lor 12 h.HC252 reumn animals鳞asep改378±Q5"C by a thermoooupie-egulated were then resuspendad in 200 Hl of 0.05%Trypan Blue solution heating lamp during schemis and reperfusion unti the animals and survMing nourons were oountod using a.To repained consciousness and re-cstabished temmohomcostasis
et al., 2000; Namura et al., 2001), MEK activation was linked to neuroprotection in ischemic tolerance and hypothermia, and in estrogen treatment against excitotoxic lesions (Hicks et al., 2000; Strohm et al., 2000; Kuroki et al., 2001). We previously reported that treating animals with Nacetyl-O-methyldopamine (NAMDA) increased the number of surviving CA1 neurons after transient global ischemia (Cho et al., 1999b). The purpose of the current study is to establish a role for ERK and its downstream CREB signaling in neuroprotection following ischemia. Specifically, we focused to investigate whether NAMDA-induced neuroprotection occurs via an ERK–CREB pathway in in vitro (oxygen glucose deprivation, OGD) and in vivo ischemic conditions. EXPERIMENTAL PROCEDURES Materials Cell culture media and antibiotics were obtained from Media Tech (Herndon, VA, USA). N2 supplement, basic fibroblast growth factor (bFGF), gentamicin, and fungizone were purchased from Gibco (Grand Island, NY, USA). Poly-L-ornithine and laminin were obtained from Sigma (St. Louis, MO, USA) and BD Bioscience (Bedford, MA, USA), respectively. NAMDA was synthesized from methyldopamine hydrochloride (Aldrich Chemical Co., Milwaukee, WI, USA) according to the method described previously by Cho et al. (1999b). The antibodies directed against phosphorylated Ser133 and phosphorylation state-independent CREB were obtained from Upstate Biotechnology (Lake Placid, NY, USA). The antibodies directed against dually phosphorylated Thr202 and 204 and phosphorylation state-independent ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). U0126 (a MEK1/2 inhibitor), SB203580 (a p38 MAPK inhibitor), LY294002 (a phosphatidylinositol 3(PI3)-kinase inhibitor) and other chemicals were obtained from Sigma. Cell cultures The immortalized neuronal progenitor cell line HC2S2 was derived from adult rat hippocampus (Hoshimaru et al., 1996) and was obtained from Dr. Fred Gage at the Salk Institute (San Diego, CA, USA). HC2S2 cells were grown in serum-free Dulbecco’s Modified Eagle medium (DMEM)/Ham’s /Ham’s F-12 (50/50) with glutamine containing N2 supplement (insulin at 5 g/ml, human transferrin at 50 g/ml, 20 nM progesterone, 100 M putrescine, 30 nM sodium selenite), bFGF at 2 ng/ml, gentamicin and fungizone on a plastic tissue culture dish coated with poly-L-ornithine and laminin. Cells were split when they were 70–80% confluent. OGD This system is used to generate an ischemic-like condition in vitro. Cultured HC2S2 cells (1105 ) were replaced with either glucose containing or glucose-free DMEM/Ham’s F-12 (50/50) and treated with either one or combination of following drugs: NAMDA (5 mM), U0126 (20 l), SB203580 (10 l) or LY294002 (10 l). The cultures were immediately placed in a chamber (Billups-Rothenberg, Del Mar, CA, USA). Except non-OGD controls, chambers containing cultures for OGD were flushed with 95% N2/5% CO2 gas mixture for 15 min at a flow rate of 4 l per minute (LPM) to create a hypoxic condition. Both normoxic and hypoxic chambers were incubated at an 37 °C incubator for 12 h. HC2S2 neurons were then resuspended in 200 l of 0.05% Trypan Blue solution and surviving neurons were counted using a hemocytometer. To examine neuronal morphology, HC2S2 neurons (1103 ) in chamber slides after 12 h OGD were fixed with 2% cold formaldehyde in 0.1 M sodium phosphate buffer (PB; pH 7.2), and stained with 0.1% Cresyl Violet. Western blot hybridization Cells (1106 ) were washed with phosphate-buffered saline (PBS; 0.1 M, pH 7.4), collected, and centrifuged to pellet. To lyse the cell pellet, 0.5 ml of RIPA buffer (1 PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)) with freshly added phenylmethyl sulphonyl fluoride (PMSF 0.4 mM), aprotinin (30 l/ml), and sodium orthovanadate (1 nM) were added to the pellet, incubated for 30 min on ice, centrifuged at 1000g for 15 min at 4 °C. Protein concentration from the supernatant was determined (Bio-Rad Laboratories, Hercules, CA, USA) and 30 g of proteins were loaded for SDS-polyacrylamide gel electrophoresis (PAGE). Electrophoresis was performed and proteins were transferred from the gel to low protein binding Durapore (PVDF) (Amersham Phamarcia Biotech Inc., Piscataway, NJ, USA) using an electroblotting apparatus. Membranes were blocked overnight in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% dry milk, incubated with primary antibodies that recognize pCREB (1:1000), pERK (1:2000), and c-Fos (1:1000; Oncogene Research Products, Cambridge, MA, USA), followed by horseradish peroxidase-conjugated secondary antibodies for 1 h. Membranes were washed three times (30 min each) with TBS containing 0.1% Tween-20 between each step. Protein bands were visualized by an ECL Plus Western blotting detection system (Amersham Pharmacia Biotech Inc.). The membrane is reblotted to visualize nonphosphorylated form of ERK (1:1000) and CREB (1:1000). For quantification, densities of phosphorylated ERK and CREB were normalized by corresponding non-phosphorylated forms of ERK and CREB. The values were then expressed in relation to that of non-OGD cultures. Transient forebrain ischemia Animal surgery was in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and The Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. All efforts were made to minimize the number of animals used and their suffering. Adult male Wistar rats (200–250 g; Hill Top, Scottsdale, AZ, USA) were anesthetized with a mixture of halothane (1%), oxygen, and nitrogen and surgically prepared for four-vessel occlusion (4-VO) according to the method described by Pulsinelli et al. (1982). Briefly, both common carotid arteries were occluded using reversible clasps and vertebral arteries were occluded by electrocauterization. Collateral blood flow to the brain was controlled by an adjustable neck suture. A burr hole on the skull (1 mm posterior to bregma, 1.5 mm lateral) was drilled for post-ischemic administration of U0126 or vehicle after ischemia. Animals were deprived of food overnight, but water was freely available. On the following day, 10 min 4-VO ischemia was induced by tightening the clasps around the common carotid arteries and the suture. To minimized variability, the following criteria were applied: loss of righting reflex and bilateral pupil dilation during the entire ischemic period and 205 min of post-ischemic coma after 10 min ischemia. Animals were treated with either triple injection of saline or NAMDA (10 mg/kg) at 0, 1/2, and 2 h after ischemia. In addition, while animals were in postischemic coma, 5 l of 100 M U0126 (total 0.5 nmoles) or vehicle (0.4% dimethyl sulphoxide, DMSO) were injected over 5 min at the lateral ventricle (3.5 mm from dura). The body temperature of all animals was kept at 37.50.5 °C by a thermocouple-regulated heating lamp during ischemia and reperfusion until the animals regained consciousness and re-established thermohomeostasis. 148 E. M. Park et al. / Neuroscience 123 (2004) 147–154
E.M Park al.Neumxciene 123 (2001)147-154 149 Tissue praparation A 0e0 Animaks were ancsthetized wth sodum pentobartical (120 mokg) NN+U N+5B N+LY nitria and 10 LUm heparin sufsne folerwd by 4%rold fermaide- pERK1 rein01MF阳pH72头The hrains ware further post-Sxed To PERK2 2h and stored in a 30%sucrose soluion ovomight Using a slding micrctome,fe dorsal hippocsmpus bctwcen bregma -3.0 mm ERK1 rd -40 mm wak anctioned a Ifirknes nl 20 um feor Crpyl ERK2 Violat slaning and 40 am or immunoeytochamistry. Immunoeytochemistry & 15 Imunceychamisry was performad using an avicdinbiotin per- m5.CA,U8】mwho对Chot.19g◆0nsh,ec知g were incabeled in 0t M PBS ennbining 3%H.C.and 10% 岳 10 seg-any incubalod avemight with one of the folowing specinc antibodics in 1%nomal scrum:phosphorylatod CREB (1:5001. phosphorylsted ERK (1:300)and c-Fos (1:10.000).On the fcl :rgcy,h的我h4mn由Hwhl州i arti-mouse Forse antisody.1:200)for 1 h.and avdinibotrper- V NN+U N+SB N+LY oxidase for 1 h in a chamber.FBS was used to wash sections betwcen al steps.The antgen antbody camolxos OGD were visuaiznd hy inra.hasan fnr 5 min in 0.05%3.3'-damineban- zicine (DAB)ar with C.1%N (Ni-DAB to anhance color rencson) Fig.1.ERK phosphorlason in HC282 neurons.A.Shown are West and 0.003%HO..To test the specifcity of ansbocics,primary m blata al ERK1/2 in lo 12 h DGD Qubure were Irealed wih NAVDA or NAMOA wih an indicated (23 mpml of anabody working sol.tion)in FBS for 2 h and then lollewnd imuocylochamical preceduras deccribed Rbeve.For were much more prono.nced.ERKC2 was used lo nommalze he den Te quantication of pCREB immunoreactvity,the mean incansi:y of immunoreactivty in the ontire CA1 pyramidal layer (moan in- were cxpteraed ira tuios cf fon-CGO curlrol cullures urd ivengod ftm fre aNe n indnpendent pcperimantx (masnD V.wahiri strala radatum as background. 1001%DVSO%N.5 mM NAMDA:U.20 HM UOt28:S,1o M SB200580:L.10 uM LY294002 PeD.05 vs.N (NAMDA)one-way Neuron density measurement analytis of varlance (ANCVA).Rsher's pmrecied least tignifrart diference (FLBD)muliple oomparison test Neuren dansity in Ihe CA1 pryramidal layer was assad aren:l ng1b中method dascribed previcusly hy Cha编d.I1abl. treatment wih U0126,a specifc MEK inhibior.Treatment Ericty.digital images of the CAt hippocampus wore aoquirod on with SB203680 (a p38 MAPK inh bitor),or LY294002 (a a Zoiss Axophoto microscope fited wth an Axdocam vidco cam- P kinase inhb tor)produced ittle repression of NAMDA induced ERK phosphorylation,indicating no cross-reactiv NY.USA)Imagea wera overaid wih a gid compesed of 100 μmX100山n bowes to facii1 sto systema6 e counting along he ity of these kinases with the MEK signaing pathway. and y-axes.Two sections at the level of dorsal hippocampus CREB phosphorylation was also assessed under the iden (bregma -3.B mm)at lcast 100 wm apart were cvalated blater- tical cultura conditians (Fig.2)Thera was a 40%reductian of pCREB exprnssion in OGD axposnd as campamd in diticns to chtain maan density fer aach anma. non-OGD control cultures (Fig.2B).Simiar to ERK phos- phorylation.treating cultures with NAMDA during OGD RESULTS reversed the reduction.In addion,the NAMDA-nduced CREB phosphorylation was aignificanlly repressed by co- ERK-induced CREB phosphorylation in treatment wth U0126.but neither by 5B203580 nor NAMDA-treated OGD neurons LY294002.The expression level of c-Fos,a target tran ERK phosphorylation in rat hippocampal-derived HC252 soription factor of CREB,was parallel with that of pCREB neurons subjected io OGD was first assessed in the pres response 1o these agants (Fig.2A1. ence and the absence of the neuroprobectant NAMDA (Fig. 1).There ware high besal lavek of phosphorylated ERK ERK-dependent neuroprotection in NAMDA-treated (pERK)and pCREB in non-OGD control cultures (Figs.1 OGD neurons and 2).probably due to the presence of bFGF in the culture To investigale whether NAMDA-induced ERK and mecia.Harwever,comparnd to non-OGD contral cultures CREB activation is invplynd in neuraprotaction,the mor- oultures exxpased to 12 h OGD shownd rnduced axpres- phalogy and the number of survving nnurons were as. sion of pERK [Fig.1).Treating cultures with NAMDA dur- sessed in HC252 neurons following OGD treatment ing OGD reversed the reduction.The NAMDA-induced (Fig.3).Compared to non-OGD control cultures (Fig ERK phosphorylation was significantty repressed by co 2A,H),exposure to 12 h af OGD resuhed in 70%neu
Tissue preparation Animals were anesthetized with sodium pentobarbital (120 mg/kg) and perfused transcardially with saline containing 0.5% sodium nitrite and 10 U/ml heparin sulfate followed by 4% cold formaldehyde in 0.1 M PB (pH 7.2). The brains were further post-fixed for 2 h and stored in a 30% sucrose solution overnight. Using a sliding microtome, the dorsal hippocampus between bregma 3.0 mm and 4.0 mm was sectioned at a thickness of 20 m for Cresyl Violet staining and 40 m for immunocytochemistry. Immunocytochemistry Immunocytochemistry was performed using an avidin/biotin peroxidase (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) method (Cho et al., 1999a). Briefly, sections were incubated in 0.1 M PBS containing 3% H2O2 and 10% methanol, followed by 2% normal horse or goat serum, and subsequently incubated overnight with one of the following specific antibodies in 1% normal serum: phosphorylated CREB (1:500), phosphorylated ERK (1:300), and c-Fos (1:10,000). On the following day, the sections were incubated with appropriate biotinylated secondary immunoglobulin G (anti-rabbit goat antibody or anti-mouse horse antibody; 1:200) for 1 h, and avidin/biotin/peroxidase for 1 h in a humidified chamber. PBS was used to wash sections between all steps. The antigen–antibody complexes were visualized by incubation for 5 min in 0.05% 3,3-diaminobenzidine (DAB) or with 0.1% Ni (Ni-DAB to enhance color reaction) and 0.003% H2O2. To test the specificity of antibodies, primary antibodies were preabsorbed with respective cognate peptides (2–3 mg/ml of antibody working solution) in PBS for 2 h and then followed immunocytochemical procedures described above. For the quantification of pCREB immunoreactivity, the mean intensity of immunoreactivity in the entire CA1 pyramidal layer (mean intensity per pixel) was obtained after subtracting mean intensity of strata radiatum as background. Neuron density measurement Neuron density in the CA1 pyramidal layer was assessed according to the method described previously by Cho et al. (1999b). Briefly, digital images of the CA1 hippocampus were acquired on a Zeiss Axiophoto microscope fitted with an Axiocam video camera, using KS400 image analysis software (Zeiss, Thornwood, NY, USA). Images were overlaid with a grid composed of 100 m100 m boxes to facilitate systematic counting along the xand y-axes. Two sections at the level of dorsal hippocampus (bregma 3.8 mm) at least 100 m apart were evaluated bilaterally by an investigator who was blinded to the experimental conditions to obtain mean density for each animal. RESULTS ERK-induced CREB phosphorylation in NAMDA-treated OGD neurons ERK phosphorylation in rat hippocampal-derived HC2S2 neurons subjected to OGD was first assessed in the presence and the absence of the neuroprotectant NAMDA (Fig. 1). There were high basal levels of phosphorylated ERK (pERK) and pCREB in non-OGD control cultures (Figs. 1 and 2), probably due to the presence of bFGF in the culture media. However, compared to non-OGD control cultures, cultures exposed to 12 h OGD showed reduced expression of pERK (Fig. 1). Treating cultures with NAMDA during OGD reversed the reduction. The NAMDA-induced ERK phosphorylation was significantly repressed by cotreatment with U0126, a specific MEK inhibitor. Treatment with SB203580 (a p38 MAPK inhibitor), or LY294002 (a PI3 kinase inhibitor) produced little repression of NAMDAinduced ERK phosphorylation, indicating no cross-reactivity of these kinases with the MEK signaling pathway. CREB phosphorylation was also assessed under the identical culture conditions (Fig. 2). There was a 40% reduction of pCREB expression in OGD exposed as compared to non-OGD control cultures (Fig. 2B). Similar to ERK phosphorylation, treating cultures with NAMDA during OGD reversed the reduction. In addition, the NAMDA-induced CREB phosphorylation was significantly repressed by cotreatment with U0126, but neither by SB203580 nor LY294002. The expression level of c-Fos, a target transcription factor of CREB, was parallel with that of pCREB response to these agents (Fig. 2A). ERK-dependent neuroprotection in NAMDA-treated OGD neurons To investigate whether NAMDA-induced ERK and CREB activation is involved in neuroprotection, the morphology and the number of surviving neurons were assessed in HC2S2 neurons following OGD treatment (Fig. 3). Compared to non-OGD control cultures (Fig. 3A, H), exposure to 12 h of OGD resulted in 70% neuFig. 1. ERK phosphorylation in HC2S2 neurons. A, Shown are Western blots of ERK1/2 in neuronal cultures exposed to 12 h OGD. Cultures were treated with NAMDA or NAMDA with an indicated inhibitor prior to subject to 12 h OGD. Note that the changes of pERK2 were much more pronounced. ERK2 was used to normalize the densities of pERK2. B, For quantification, the optical density (OD) values were expressed as ratios of non-OGD control cultures and averaged from five sets of independent experiments (meanS.D.). V, vehicle (0.01% DMSO); N, 5 mM NAMDA; U, 20 M U0126; S, 10 M SB203580; L, 10 M LY294002. * P0.05 vs. N (NAMDA), one-way analysis of variance (ANOVA), Fisher’s protected least significant difference (PLSD) multiple comparison test. E. M. Park et al. / Neuroscience 123 (2004) 147–154 149
150 E.M.Park al.Neurescianea 123 (2004)147-154 A OGD N NU N+SB N+LY DCREB CREB oFOS B 15 H 120 、、 100 40 20 V NN+U N+SB N+LY VN N+U N+88 N+LY OGD OGD Fig.2.CRB phnephnryarion in HC2 neumns.A.Shoan am Westom blals of pCREB and CREB.and c-Fos in neuronal cultures Flg.3.ERK-deperdem neuroprotecton againet OGD.HC252 neu- eapooed to 12 h O3O.Cuurea were temrod wih NAMDA or NAMDA mrw和td512h0GDw时情t中nwh5hn减的mne their morphology.A.Non-OGD oonrot B.OGD:C.0GD+5 ml d如aal物上箱ars titt创pG任台.BF年Ilt输 NANDA (NE D.OGD+N+20 WM U0126 (U%E CGD+N+10 M 8E20359019EF00D+N+10uMLY294002LM:G,20.MU0128 w特可对tsm4g时pendaml cxp6n--t杯TsnE5D)V vchicle 10.01%DMSOL N.5 mM NAMDA:U.20 uM UOt26:S.10 pM nairen.HC:.neuions wer trroled wih NANDA n NAMDA wih 5B2358路L.10MLY29402.'P<0.85gV【eel.Pc0.06 a.N (NAMDA线one-wiry ANOVA.Fiah起r幽PL3Dul年出Gem中aiam sunvMing neurons was counted at the end ol DGD.Values represert mean=8.D.Pc0.05 va.V [).Pe0.01 va.N one-way ANOVA Fisher's PLSD muliple comrpsrison lest (n-5-7]Imoga ronal death (Fig.3B).NAMDA significantly increased the 3动6p,r50m60 number of surviing neurons (Fig.3C:P0.05).The NAMDA-nduced neuroprotection disappeered wth criens and stratum radiatum s assocated wth CREB ac U0126 co-treatment [Fig.3D;P20.01).but not with tivation in NAMDA-treated CA1 sub-region,CREB pho8- SB203580-or LY294002-trealed cultures.The morphol- agy of UD126-treated CGD cultures was not dierent phorylstion was assessed in ischemic hppocampus (Fig. from that of CGD culture shown in Fig.3B.In addition, 4Ad-f)pCREB poeitive CA1 pyramidal neurors were neurons retained normal morphology when the 8ame rarely found in the control hippocampus.At 3 h post- concentration of U0126 was added to non-OGD-treated schemia,DCREB immunoractivity in the CA1 pyramidel cultures (Fig.3G),ind caling the absence of non-specific layer was slightly increased along with a few pCREB pos- cytotoxicity of UD126 81 the concentration used. itie naurons (Fig 4An).Tmatment of NAMDA at D.1/2 and 2 h of reperfusion significantly increasnd pCREB im- NAMDA-induced ERK/CREB phosphorylation in the munoreaclivily in the CA1 pyramidal layer (Fig.4A f. arly post-lschemia hippocampus P.c0.05).Preabeorption with respective cognale peptides Immunolocalzation of pERK during earty reperfusion to pERK and pCREB pnmary antiserum completely abal hours was examined in rat hippocampus afer 10 min of ished the staining of pERK and pCREB in the adjacent 4-VD ischamia.In the contrl and 3 h posst ischemin hip- sections (data not shown) pocampus,pERK immunoreactity (Fig 4Aa,b)was ob We further excamined the expression of o-Fos.a prutein served in sratum oriens and stratum radiatum,but absent product enooding CRE-dependent cfos gene,to confirm in the pyramidal layer of the CA1 sub-regian.The immu that CREB activation leads to the transcription of target noreactmvty of pERK in the stratum crens and stratum genes.Ischemia caused a sight induction of c-Fos in the radistum was increased in the animal freated with NAMDA CA1 pyramidal lsyer 6 h after ischemia (Fig.4Bb)and the at 0.1/2 and 2 h of reperfusion (Fig.4Ac).n order to expression was farther potentiated in the NAMDA-treated determine whether increased pERK activty in the stratum CA1 neurons (Fig.4Bc)
ronal death (Fig. 3B). NAMDA significantly increased the number of surviving neurons (Fig. 3C; P0.05). The NAMDA-induced neuroprotection disappeared with U0126 co-treatment (Fig. 3D; P0.01), but not with SB203580-or LY294002-treated cultures. The morphology of U0126-treated OGD cultures was not different from that of OGD culture shown in Fig. 3B. In addition, neurons retained normal morphology when the same concentration of U0126 was added to non-OGD-treated cultures (Fig. 3G), indicating the absence of non-specific cytotoxicity of U0126 at the concentration used. NAMDA-induced ERK/CREB phosphorylation in the early post-ischemic hippocampus Immunolocalization of pERK during early reperfusion hours was examined in rat hippocampus after 10 min of 4-VO ischemia. In the control and 3 h post ischemic hippocampus, pERK immunoreactivity (Fig. 4Aa, b) was observed in stratum oriens and stratum radiatum, but absent in the pyramidal layer of the CA1 sub-region. The immunoreactivity of pERK in the stratum oriens and stratum radiatum was increased in the animal treated with NAMDA at 0, 1/2 and 2 h of reperfusion (Fig. 4Ac). In order to determine whether increased pERK activity in the stratum oriens and stratum radiatum is associated with CREB activation in NAMDA-treated CA1 sub-region, CREB phosphorylation was assessed in ischemic hippocampus (Fig. 4Ad–f). pCREB positive CA1 pyramidal neurons were rarely found in the control hippocampus. At 3 h postischemia, pCREB immunoreactivity in the CA1 pyramidal layer was slightly increased along with a few pCREB positive neurons (Fig. 4Ae). Treatment of NAMDA at 0, 1/2 and 2 h of reperfusion significantly increased pCREB immunoreactivity in the CA1 pyramidal layer (Fig. 4A f, g; P0.05). Preabsorption with respective cognate peptides to pERK and pCREB primary antiserum completely abolished the staining of pERK and pCREB in the adjacent sections (data not shown). We further examined the expression of c-Fos, a protein product encoding CRE-dependent c-fos gene, to confirm that CREB activation leads to the transcription of target genes. Ischemia caused a slight induction of c-Fos in the CA1 pyramidal layer 6 h after ischemia (Fig. 4Bb) and the expression was further potentiated in the NAMDA-treated CA1 neurons (Fig. 4Bc). Fig. 2. CREB phosphorylation in HC2S2 neurons. A, Shown are Western blots of pCREB and CREB, and c-Fos in neuronal cultures exposed to 12 h OGD. Cultures were treated with NAMDA or NAMDA with an indicated inhibitor prior to subject to 12 h OGD. CREB was used to normalize the densities of pCREB. B, For quantification, the OD values were expressed as ratios of non-OGD control cultures and averaged from five sets of independent experiments (meanS.D.). V, vehicle (0.01% DMSO); N, 5 mM NAMDA; U, 20 M U0126; S, 10 M SB203580; L, 10 M LY294002. * P0.05 vs. V (vehicle), # P0.05 vs. N (NAMDA), one-way ANOVA, Fisher’s PLSD multiple comparison test. Fig. 3. ERK-dependent neuroprotection against OGD. HC2S2 neurons subjected to 12 h OGD were stained with Cresyl Violet to examine their morphology. A, Non-OGD control; B, OGD; C, OGD5 mM NAMDA (N); D, OGDN20 M U0126 (U); E, OGDN10 M SB203580 (SB); F, OGDN10 M LY294002 (LY); G, 20 M U0126 without OGD; H, quantification of the number of surviving HC2S2 neurons. HC2S2 neurons were treated with NAMDA or NAMDA with an indicated inhibitor prior to subject to 12 h OGD. The number of surviving neurons was counted at the end of OGD. Values represent meanS.D. * P0.05 vs. V (vehicle), # P0.01 vs. N, one-way ANOVA, Fisher’s PLSD multiple comparison test (n5–7). Images were adjusted for optimal contrast and sharpening using Adobe photoshop, version 6.0. 150 E. M. Park et al. / Neuroscience 123 (2004) 147–154
E.M.Park al.Neresciane 123 (2004)147-154 151 Cortrol Echemia+V ischema+N N N+U PERK Cresyl violet 日 A 2 Cont N N+U ① Codl Ischoma+V lachemia+N Fig 5.I18C-dapardent nwimemardinn ngsiro Ira-sier gnbo irh- Violet slsiring in CAt brper of vehicde-irasind (V) cHG路 NANDA-teated [NI and NAMDA+U0126-trested (N U]ischemic hippocampus measured 7 days postischemia.Triple ip.injection of fg.4 mmunineslzarion ol pe,pC旺自ndc-fes in proHarh sainn or NAMCA (10 mgng anch ima)mena givan a!D.1/2 and 2hof emic CA hippocampus.Comparable secsons at the level of corsal reperfusion.U0128(05 nmolus)wus giwen itnericularly 15 min t内ceriion kwumn darei感nimher6 nNime1G2mr4 (Ad-n.and o-Fos (8o-s)ot 3 h post-iachemia for pERK and pCREB, and A h poot-isthamis for o-Fes.Each ancon D me is nenraenlaliv from Hre auts of puired suine-and NAMCA-nslud version e.0. ity in the CA1 pyamidal layers.'Pe0.05 va.control (Cont"Pe0.06 s.m oneway ANOVA,Fisher's PLSD mutiple compa pathetis and cerebellar granufar neurans (Edwards et al.. ison tura!(n-3-4).Triple i.p.ijudlion af adine (or NAMDA (N:10 1901:Chang et 8l..1996;Chang and Korolev.1997). Furthemore,elevated Ser133 phcsphorylation of CREB CA1 pyrammital byw:SD,Rranim erian:SR xtshm racuatum Im- protects neurons against apoplosis induced by ckadsic ages were odjusted for optimal oomrast and sharpering using Adote acid (Woodgate et 1999).Nauroprotactiva mle af photoshop,version 6.0. CREB has bean dearty dalnaated i vitro and i wm stucies (Bonni el al.,1999:Riccio et al.,1999;Mabucchi et ERK-dependent neuroprotection in NAMDA-reated al.,2001;Mantamadiotis et al.,2002).Consistent with ischemic CA1 ncurons these reports,we also showed that increasing CREB phas To investigatn whether ERK actiation is acpount for neu- phorylation by a neuroprotective measure (e.g.NAMDA) ronal survival in wve,we asseased the number of sunviving was associated with increased survival of HC252 neurons neurons in animals treated with saline.NAMDA and subjected to OGD.In an experimental animal model of NAMDA+U0128.Neurcral sunvival was sgnfcanty stroie.CREB phosphory aton was shown to be 8ssoci greater in NAMDA-as compared to saine-treated isch ated wth surviving neumns hoth in glhal and focal isch- amic animals &s incicated by neuronal densiry measured 7 ema (Walon et al.,1996:Hu et al.,1999.Tanaka et al. days afer ischemia (Fig.5).The NAMDA-induced neuro- 1999,2000:V3 on and Dragnow,2000Tan33,20011. prolection was abolished by U0128 co-treatment Similar tn these resuits,we also ohsarved more CREB phosphoryation in the resistant CA3 neurons and dentale DISCUSSION granular cels compared to vulnerable CA1 in the transient global ischemic rat model (data not shown).In add tion,our The current study was directed to investgate whether ERK findings of increased neuronal survial with the increased phosphorylation and ita downstream target CREB cascade number of pCREB positive neurons in the CA1 of NAMDA- serve as an endogenous neuroprotective signaing path- treated ischemic animals support the view that CREB way in NAMDA-induced neuroprotection followng isch phosphorylation is associsled with neuroprotection after amiA.We damonstrated that ERK-dapandant CREB phos- schemia i w. phorylation and CREB-medated c-Fos axpresssion oocurs MEK phosphorylates CREB via activation of sequandal in NAMDA-treated ischemic neurons and NAMDA-induced ERK and RSK-2.Unlike the delinealed neuroprotective nnuropratectian following ischemia is ERX-dopandont. ale of CRE日，a roln of ERK is dehatable.Alnssandrini nt cAMP and ts analogue via CAMP-dependent pathway al.(1959)and Namura et alL (2001)reportod that the are known to promote survival and neurile outgrowth in inhibition of MEK1 protein kinase (hus inhibiting ERK ac- sympathetic and sensory neurons (Rydel and Greene. tivation)reduced damsge in focal ischemic animal models. 1988)and to suppress programmed cell death in rat sym However,there are many reports that support the notion
ERK-dependent neuroprotection in NAMDA-treated ischemic CA1 neurons To investigate whether ERK activation is account for neuronal survival in vivo, we assessed the number of surviving neurons in animals treated with saline, NAMDA, and NAMDAU0126. Neuronal survival was significantly greater in NAMDA- as compared to saline-treated ischemic animals as indicated by neuronal density measured 7 days after ischemia (Fig. 5). The NAMDA-induced neuroprotection was abolished by U0126 co-treatment. DISCUSSION The current study was directed to investigate whether ERK phosphorylation and its downstream target CREB cascade serve as an endogenous neuroprotective signaling pathway in NAMDA-induced neuroprotection following ischemia. We demonstrated that ERK-dependent CREB phosphorylation and CREB-mediated c-Fos expression occurs in NAMDA-treated ischemic neurons and NAMDA-induced neuroprotection following ischemia is ERK-dependent. cAMP and its analogue via cAMP-dependent pathway are known to promote survival and neurite outgrowth in sympathetic and sensory neurons (Rydel and Greene, 1988) and to suppress programmed cell death in rat sympathetic and cerebellar granular neurons (Edwards et al., 1991; Chang et al., 1996; Chang and Korolev, 1997). Furthermore, elevated Ser133 phosphorylation of CREB protects neurons against apoptosis induced by okadaic acid (Woodgate et al., 1999). Neuroprotective role of CREB has been clearly delineated in vitro and in vivo studies (Bonni et al., 1999; Riccio et al., 1999; Mabucchi et al., 2001; Mantamadiotis et al., 2002). Consistent with these reports, we also showed that increasing CREB phosphorylation by a neuroprotective measure (e.g. NAMDA) was associated with increased survival of HC2S2 neurons subjected to OGD. In an experimental animal model of stroke, CREB phosphorylation was shown to be associated with surviving neurons both in global and focal ischemia (Walton et al., 1996; Hu et al., 1999; Tanaka et al., 1999, 2000; Walton and Dragnow, 2000; Tanaka, 2001). Similar to these results, we also observed more CREB phosphorylation in the resistant CA3 neurons and dentate granular cells compared to vulnerable CA1 in the transient global ischemic rat model (data not shown). In addition, our findings of increased neuronal survival with the increased number of pCREB positive neurons in the CA1 of NAMDAtreated ischemic animals support the view that CREB phosphorylation is associated with neuroprotection after ischemia in vivo. MEK phosphorylates CREB via activation of sequential ERK and RSK-2. Unlike the delineated neuroprotective role of CREB, a role of ERK is debatable. Alessandrini et al. (1999) and Namura et al. (2001) reported that the inhibition of MEK1 protein kinase (thus inhibiting ERK activation) reduced damage in focal ischemic animal models. However, there are many reports that support the notion Fig. 4. Immunolocalization of pERK, pCREB and c-Fos in post-ischemic CA1 hippocampus. Comparable sections at the level of dorsal hippocampus of CA1 are used to localize pERK (Aa–c), pCREB (Ad–f), and c-Fos (Ba–c) at 3 h post-ischemia for pERK and pCREB, and 6 h post-ischemia for c-Fos immunohistochemistry. Each section is representative from three sets of paired saline- and NAMDA-treated animals after ischemia. B, g, Quantification of pCREB immunoreactivity in the CA1 pyramidal layers. * P0.05 vs. control (Cont), # P0.05 vs. ischemia (Ish), one-way ANOVA, Fisher’s PLSD multiple comparison test (n3–4). Triple i.p. injection of saline (V) or NAMDA (N; 10 mg/kg each time) were given at 0, 1/2 and 2 h of reperfusion. CA1, CA1 pyramidal layer; SO, stratum oriens; SR, stratum radiatum. Images were adjusted for optimal contrast and sharpening using Adobe photoshop, version 6.0. Fig. 5. ERK-dependent neuroprotection against transient global ischemia. Cresyl Violet staining in CA1 layer of vehicle-treated (V), NAMDA-treated (N) and NAMDAU0126-treated (N U) ischemic animals 7 days after ischemia. Mean neuron density in the CA1 hippocampus measured 7 days post-ischemia. Triple i.p. injection of saline or NAMDA (10 mg/kg each time) were given at 0, 1/2 and 2 h of reperfusion. U0126 (0.5 nmoles) was given intraventricularly 15 min after reperfusion. Neuron densities (number of neurons/105 m3 ) are expressed as meanS.D. N5–7 for each group. * P0.05 vs. V, # P0.05 vs. N, ANOVA, post-hoc Fisher’s PLSD. Images were adjusted for optimal contrast and sharpening using Adobe photoshop, version 6.0. E. M. Park et al. / Neuroscience 123 (2004) 147–154 151
15知 E.M Park al.Neumxciene 123 (2001)147-154 that ERK Activation is a neumpmtective signal (OAwA at indicatas that ERK-CREB s gnaling pethwary might he in- al.,1999;Shamlo et aL,1999:Hicks et al.,2000;Strohm wolved in the survival of NAMDA-treated ischemic neurons et al..2000).In ischemic animals,neuroprolective mea- Ischemis allered expression of a number of genes sures such as precandtioning and hypothermia were incuding mmediate eary genes heat shack protein,basic inked lo ERK activation (Hicks et al.,2000;Strohm et al.. FGF,nerve growth factor,brain-denved neurotrophic fao 2000).ERK activation also protoctod neurons against tors.and mamy other trophic factors and receptors (Fin- quindlinate and glutamate xcity (Singer et al.,1999; klestein et al.,1988,1990:Lindvall et al..1992;Nowak Kuroki et al.,2001:see review by Lee and McEwen.2001). 1993;Takeda et al.,1993:Hsu et al.,1993,1994),some of Furthermore.urocortin,a member of the corticotorpin re which are implicated in functional recovary after stroke easing hormone family of neuropepbdes,has been shown Phosphorylation of a critical CREB residua,SerM33.re- n Bctivate ERK 1/2 and to protect cultured hippocampal sults in transsclivabon of some larget genes including neurons from oxidalive and exctoloxic cell death [Gram c-as,bci2,bdf Mol,as wall as many ather unidantified matopoules et aL 2000:Pedersen et al.,2002).The dis genas.Prolongatian of CRE binding arvity via ERK,thus, orepanmy betwnen neuroproboctivn and advnrse nffects af may be one of the crical steps neceesary to prevent ERK activation may lie in either difference in experimental ischemia-induced neuronal injury.Supporting this idea is animal models or dual potential of ERK adtvaticn.Ish the unigue action of NAMDA that increases expression af kawa and Kitamura (1999)reported that inhibit on of tran activated ERK and CREB and subsequently protedted sient up-regulation of ERK procects neurona from H.O neurons from insuts both in wo and in wvo.In addition. induced cell death.In contrast,the inhibition of prolnga increased CREB phosphorylation was followed by in- tion of basal and constitutive activity of ERK resuled in cell creased the number of c-Fos positive neurons in NAMDA- daath.Since ERK Activation ooours within minutas of treated ischamic CA1 neurons t aarly reperfusion hours reperfusion after ischemia,inhibition of insul-induced tran (Fig.48),indicating that CREB phasphoryabion leads 1n sient ERK activation may afford neuroprotaction as re- the transcripon of c-fos,a downstream CREB-medialed porind by Alessandrini (1999)and Namura at al.(2001). target gene.The fnding supports a funstional role of in. On the other hand.applying neuroprolective measures creased CREB activation in wvo.The previously reported a [i.e.pre-ischemi condtoning,hypothemia,and astro possible neuroprotective role of early c-Fos induction after gen)may increase or prolong ERK amtivation,which sub cerebral ischemia (Cho et 8l..2001)is consistant with our sequenty turns on survival cascades.Stanciu and De- present fnding that CRE-dependent gene transcription Franco (2002)recentty repcrted that prolonged nudear may be required for nauronal survival. retention of actvated ERK is a critical factor in aliciting In Summary.the current shuady damonstrates the mle of proapoptatic effects in neurons suhjected to oxidative ERK in hippocampal neuronal survival and provides a segs.Hlowever,the3u出cr形were unable to link between basis lo develop therapeutic stralegies aimed at augment- nuclear ratention of ERK and CREB phosphorylation in the ing the ERK-CREB cascada to nnhancn nouronal survival study,suggasting the prasancn of downstream avants against pathological insuts such as ischemia. other than CREB phosphorylation afler ERK aclivaton Theredore,t is tempting to speculate that endogenous Ani0 mloogcmon的-Gm0请W话40在d by AHA to&C surviving cascades such as a ERK-CREB pathway can be and Hurke Foundaton. enhanced by neuroprotective measure (or preconditioning) and that ERK activation caused by insut alne or insult REFERENCES with neuroprocective measures may ditferentially activate downstream-signeling pathways.Unlie the sbave studies cmain kinsse inhitison protedds灯n对Ganj5 eailting f■ that focused only on an ERK signaling pathway with neu- ronal daath/survival.the prasant study was diracted t 上sGM&khJC.Pral脑山，Tak」M含a由JD1g dafne the roka of ERK signaing in melation to CREB acti vation.Therefore.NAMDA-induced neuroprotedtion vis r.Nm12-9 forni A.fnie:A.Wrd Af,Dstts SR Takxu MA,Gmense'g MP ERK activation that leads to CREB phosphorylation during a critical period maybe a key step toward transcription of Ey trarerpson-dpendunl and Andipendert mechinima.So- CRE-dependent genes necessary for Burvival. er6w288.1358-1352 Mechanistic studies using an in wtro ischemic condi tion revealed that the inhibition of other P38 MAPK did not a脚rn可-artivating polypept恤PACAP)prevrt pm- l's.Nro affect the NAMDA-induced ERK-CREB signaling pathway aL41208181-184 and neuroprulection,indicatng no croes-inhibition of ERK activaton by the P38 inhbitor.In ard tion,the inhbition af pmgrammns cal dasth Nwuimcham Int 31:161-167 the protein khnase B pathway had Itte effect on ERK- Ona8 Hwang Bakar H,BakH,中8T.8an山，JnTH(ie dependen CREB phosphorylation and on the number of surviving neurons in NAMDA-treated cultures.On the dopamne i the pinedl dand 8yrupse 34.185-144. Cho S,Volge BT,Bae Y,Hwang O.Choi HJ,GalJ,Park LCH,ChaCK other hand,the inhibition of ERK activation by U0125 口udn的TH(i级h句hk:精rahrydimbiopelrin xyrheo对n attenuated NAMDA-induoed CREB activation and the sur- prbacta naurora abr traroien forbrain bachmia的ta它wl vival of hippucampal neurorss follwing OGD.The fnding rek for the cofuctor.J Neuresci 19.878-850
that ERK activation is a neuroprotective signal (Ozawa et al., 1999; Shamloo et al., 1999; Hicks et al., 2000; Strohm et al., 2000). In ischemic animals, neuroprotective measures such as preconditioning and hypothermia were linked to ERK activation (Hicks et al., 2000; Strohm et al., 2000). ERK activation also protected neurons against quinolinate and glutamate toxicity (Singer et al., 1999; Kuroki et al., 2001; see review by Lee and McEwen, 2001). Furthermore, urocortin, a member of the corticotorpin releasing hormone family of neuropeptides, has been shown to activate ERK 1/2 and to protect cultured hippocampal neurons from oxidative and excitotoxic cell death (Grammatopoulos et al., 2000; Pedersen et al., 2002). The discrepancy between neuroprotective and adverse effects of ERK activation may lie in either difference in experimental animal models or dual potential of ERK activation. Ishikawa and Kitamura (1999) reported that inhibition of transient up-regulation of ERK protects neurons from H2O2- induced cell death. In contrast, the inhibition of prolongation of basal and constitutive activity of ERK resulted in cell death. Since ERK activation occurs within minutes of reperfusion after ischemia, inhibition of insult-induced transient ERK activation may afford neuroprotection as reported by Alessandrini (1999) and Namura et al. (2001). On the other hand, applying neuroprotective measures (i.e. pre-ischemic conditioning, hypothermia, and estrogen) may increase or prolong ERK activation, which subsequently turns on survival cascades. Stanciu and DeFranco (2002) recently reported that prolonged nuclear retention of activated ERK is a critical factor in eliciting proapoptotic effects in neurons subjected to oxidative stress. However, the authors were unable to link between nuclear retention of ERK and CREB phosphorylation in the study, suggesting the presence of downstream events other than CREB phosphorylation after ERK activation. Therefore, it is tempting to speculate that endogenous surviving cascades such as a ERK–CREB pathway can be enhanced by neuroprotective measure (or preconditioning) and that ERK activation caused by insult alone or insult with neuroprotective measures may differentially activate downstream-signaling pathways. Unlike the above studies that focused only on an ERK signaling pathway with neuronal death/survival, the present study was directed to define the role of ERK signaling in relation to CREB activation. Therefore, NAMDA-induced neuroprotection via ERK activation that leads to CREB phosphorylation during a critical period maybe a key step toward transcription of CRE-dependent genes necessary for survival. Mechanistic studies using an in vitro ischemic condition revealed that the inhibition of other P38 MAPK did not affect the NAMDA-induced ERK–CREB signaling pathway and neuroprotection, indicating no cross-inhibition of ERK activation by the P38 inhibitor. In addition, the inhibition of the protein kinase B pathway had little effect on ERKdependent CREB phosphorylation and on the number of surviving neurons in NAMDA-treated cultures. On the other hand, the inhibition of ERK activation by U0126 attenuated NAMDA-induced CREB activation and the survival of hippocampal neurons following OGD. The finding indicates that ERK–CREB signaling pathway might be involved in the survival of NAMDA-treated ischemic neurons. Ischemia altered expression of a number of genes including immediate early genes, heat shock protein, basic FGF, nerve growth factor, brain-derived neurotrophic factors, and many other trophic factors and receptors (Finklestein et al., 1988, 1990; Lindvall et al., 1992; Nowak 1993; Takeda et al., 1993; Hsu et al., 1993, 1994), some of which are implicated in functional recovery after stroke. Phosphorylation of a critical CREB residue, Ser/133, results in transactivation of some target genes including c-fos, bcl2, bdnf, Mcll, as well as many other unidentified genes. Prolongation of CRE binding activity via ERK, thus, may be one of the critical steps necessary to prevent ischemia-induced neuronal injury. Supporting this idea is the unique action of NAMDA that increases expression of activated ERK and CREB and subsequently protected neurons from insults both in vitro and in vivo. In addition, increased CREB phosphorylation was followed by increased the number of c-Fos positive neurons in NAMDAtreated ischemic CA1 neurons at early reperfusion hours (Fig. 4B), indicating that CREB phosphorylation leads to the transcription of c-fos, a downstream CREB-mediated target gene. The finding supports a functional role of increased CREB activation in vivo. The previously reported a possible neuroprotective role of early c-Fos induction after cerebral ischemia (Cho et al., 2001) is consistent with our present finding that CRE-dependent gene transcription may be required for neuronal survival. In summary, the current study demonstrates the role of ERK in hippocampal neuronal survival and provides a basis to develop therapeutic strategies aimed at augmenting the ERK–CREB cascade to enhance neuronal survival against pathological insults such as ischemia. Acknowledgements—This work was supported by AHA to S.C. and Burke Foundation. 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