Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

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1 (22), 2 & 22 Mcmilln Pulishers Limited All rights reserved 35947/2 Twostep ctivtion of FOXO3 y AMPK genertes coherent feedforwrd loop determining excitotoxic cell fte D Dvil, NMC Connolly 2, H Bonner, P Weisová,3, H Dussmnn,2, CG Concnnon,2, HJ Huer 2 nd JHM Prehn,,2 Cererl ischemi nd excitotoxic injury induce trnsient or permnent ioenergetic filure, nd my result in neuronl poptosis or necrosis. We hve previously shown tht ATP depletion nd ctivtion of AMPctivted protein kinse (AMPK) during excitotoxic injury induces neuronl poptosis y trnscription of the propoptotic BH3only protein, Bim. AMPK, however, lso exerts prosurvivl functions in neurons. The moleculr switches tht determine these differentil outcomes re not well understood. Using n pproch comining iochemistry, singlecell imging nd computtionl modeling, we here demonstrte tht excitotoxic injury ctivted the im promoter in FOXO3dependent mnner. The ctivtion of AMPK reduced AKT ctivtion, nd led to dephosphoryltion nd nucler trnsloction of FOXO3. Susequent muttion studies indicted tht im gene ctivtion during excitotoxic injury required direct FOXO3 phosphoryltion y AMPK in the nucleus s second ctivtion step. Inhiition of this phosphoryltion prevented Bim expression nd protected neurons ginst excitotoxic nd oxygen/glucose deprivtioninduced injury. Systems nlysis nd computtionl modeling reveled tht these two ctivtion steps defined coherent feedforwrd loop; network motif cple of filtering ny effects of shortterm AMPK ctivtion on im gene induction. This my prevent unwnted AMPKmedited Bim expression nd poptosis during trnsient or physiologicl ioenergetic stress. dvnce online puliction, 27 April 22; doi:.38/cdd Excitotoxicity hs een implicted in the pthogenesis of cererl ischemi nd severl neurodegenertive disorders, 3 nd results from norml levels of the excittory neurotrnsmitter glutmte (GLUT) in the synptic cleft. 46 The extent of ATP depletion during excitotoxic injury criticlly determines neuronl outcome. 7 Neurons tht fil to restore their ATP levels undergo rpid excitotoxic necrosis. 7 However, mny of the neurons tht initilly overcome the energetic crisis nd recover their ATP levels nevertheless undergo delyed poptotic deth. 8 ATP depletion ctivtes the AMPctivted protein kinse (AMPK), which dpts the cellulr response to ioenergetic stress. 9 AMPK switches off nolic pthwys, preventing ATP consumption, nd switches on ctolic pthwys, incresing glucose uptke nd ATP production. AMPKdependent plsm memrne trnsloction of the glucose trnsporter 3, nd susequent glucose uptke medite tolernce of neurons to excitotoxic or ischemic injury.,2 In contrst to these cytoprotective mechnisms, however, prolonged AMPK ctivtion hs lso een shown to contriute to ischemic injury nd excitotoxicity. 9,3,4 We recently demonstrted tht prolonged ctivtion of AMPK medites excitotoxic poptosis y inducing the expression of the propoptotic Bcl2 fmily protein Bim. 3 However, the mechnisms tht AMPK utilizes to switch on the trnscriptionl ctivtion of im during excitotoxic poptosis remin unknown. Here, we descrie the signling pthwys tht couple AMPK ctivtion to im gene expression during excitotoxic poptosis, nd provide mthemticl frmework tht explins cell fte decisionmking during AMPK ctivtion. Results Bim induction during excitotoxic poptosis requires AP nd FOXO3. We hve previously shown tht rief stimultion of NMDA receptors in corticl neurons or cereellr grnule neurons (CGNs) induces imdependent, delyed excitotoxic cell deth chrcteristic of cspseindependent poptosis.,3,5 Western lot (Figure ) nd qpcr nlysis confirmed tht CGNs exposed to GLUT/ glycine (GLY) (/ mm) showed elevted Bim levels. Centre for the Study of Neurologicl Disorders, Royl College of Surgeons in Irelnd, 23 St Stephen s Green, Dulin, Irelnd nd 2 Deprtment of Physiology nd Medicl Physics, Centre for Systems Medicine, Royl College of Surgeons in Irelnd, 23 St Stephen s Green, Dulin, Irelnd Corresponding uthor: JHM Prehn, Deprtment of Physiology nd Medicl Physics, Royl College of Surgeons in Irelnd, 23 St Stephen s Green, Dulin 2, Irelnd. Tel: ; Fx: ; Emil: prehn@rcsi.ie 3 Present ddress: Mx F Perutz Lortories, University of Vienn, Dr BohrGsse 9, 3 Vienn, Austri Keywords: excitotoxicity; energy depletion; BH3only protein BIM; FOXO3; computtionl modeling; network motifs Arevitions: AKTCA, constitutively ctive mutnt of AKT; AMPKCA, constitutively ctive mutnt of AMPK; CC, compound C; CFL, coherent feedforwrd loop; CGNs, cereellr grnule neurons; 6 DBE FOXO3 promoter, reporter luciferse plsmid with six copies of the FOXO fmily proteininding element; DNFOXO3, dominntnegtive FOXO3; FOXO36A, construct with FOXO3 sequence mutted t the AMPK phosphoryltion sites; FOXO3nucler, construct with FOXO3 sequence mutted t the AKT phosphoryltion sites; GLY, glycine; GLUT, glutmte; OGD, oxygen/glucose deprivtion; PI, propidium iodide; RLUs, reltive luciferse counts Received 3.9.; revised ; ccepted ; Edited y JM Hrdwick

2 Luciferse reporter ctivity (Fold increse) 2 FOXO3 nd AMPK 3 2 Bim the FOXO3 nd AP trnscription fctors, which hve een implicted in Bim expression during neuronl poptosis. 3,6 8 We trnsfected CGNs with reporter construct ering the wildtype (wt) im promoter sequence, or im promoter constructs hroring muttions in the FOXO3 or AP inding sites. Luciferse ctivity ws significntly incresed in GLUTtreted neurons expressing the wt im promoter. However, this effect ws rogted y either im promoter muttion (Figure c), indicting tht FOXO3 nd AP inding sites were necessry for im promoter ctivtion. Shm % Pyknotic nuclei c Glut / Gly (/ μm) Bim promoter Wild Type Bim promoter FOXO3 site mutted Bim promoter AP site mutted 3 2 h 4 h 8 h 6 h Time fter glutmte tretment WT im / Shm Shm Excitotoxic injury ws significntly reduced in CGNs deficient in im (Figure ). We next ddressed the signling pthwys involved in im ctivtion during excitotoxic injury. Excellent cndidtes re 24 h βactin WT im / Figure Bim induction during excitotoxic poptosis requires AP nd FOXO3. () Western lot nlysis showed significnt increse in Bim levels within 4 24 h time frme fter GLUT/GLY ( mm/ mm, 3 min) exposure (Po.5; n ¼ 5). ctin served s loding control. () CGNs from im / mice nd wt controls were treted with GLUT or experimentl uffer (shm conditions). 24 h fter tretment the neurons were stined live with Hoechst nd pyknotic nuclei scored (Po.5; n ¼ 3). Br, 2.5 mm. (c) CGNs were trnsfected with vector contining.8 kb frgment of the im promoter. Bim promoter ctivtion ws significntly incresed 24 h fter GLUT exposure (Po.5; n ¼ 3). Muttions of the FOXO3 nd AP inding sites significntly reduced this ctivtion (Po.5; n ¼ 3) AMPKdependent downregultion of the mtor/akt pthwy during excitotoxicity. Previously, we demonstrted tht pampk ctivted JNKs, with JNK ctivtion contriuting to im expression nd poptosis. 3 We therefore next focused on exploring the role of FOXO3 ctivtion in im gene induction, nd its control y AMPK. Firstly, we nlyzed AMPK ctivtion during excitotoxicity y western lot nlysis of pampk (Thr72) levels. AMPK ctivtion ws evident min fter GLUT exposure, nd recovered to seline levels fter 2 min (Figure 2). The mtor kinse complex (mtorc) is negtively regulted y AMPK. 9 To ddress the regultion of mtorc during excitotoxicity, we monitored phosphomtor (Ser2448) levels (Figure 2). GLUT tretment decresed pmtor levels min fter exposure, coinciding with the pek of AMPK ctivtion. Notly, pmtor levels were persistently downregulted in response to GLUT. mtorc2 hs een descried s n importnt ctivtor of the prosurvivl kinse AKT. 2 We detected n erly decrese in pakt (Ser473) levels in response to GLUT (Figure 2c). In contrst to pmtor, however, pakt levels prtilly recovered 2 8 h fter GLUT exposure with complete recovery fter 6/24 h. The inctivtion of AKT enles FOXO3dependent gene trnscription y preventing the cytosolic trnsloction of FOXO3. 2 Western lot nlysis of phosphofoxo3 (Thr32) levels showed significnt decrese 2 4 h fter GLUT exposure (Figure 2d). To demonstrte tht AMPK ctivtion ws responsile for the decrese in pakt levels during excitotoxic injury, we depleted AMPK levels with vectors expressing n sirna trgeting AMPK/2, or control sequence 3 (Supplementry Figure ). CGNs with depleted AMPK levels presented significntly higher levels of phosphoakt (Ser473) compred with controltrnsfected neurons (Figure 2e). This result ws confirmed using the phrmcologicl inhiitor of AMPK, compound C (CC). CGNs pretreted with CC ( mm) 45 min efore GLUT exposure presented with 69.5±9.5% higher phosphoakt (Ser473) levels 3 min fter GLUT tretment, compred with those pretreted with vehicle (dt not shown). AMPK/2 depletion lso incresed phosphofoxo3 (Thr32) levels in neurons exposed to GLUT (Figure 2e). Confirming the downregultion of FOXO3 phosphoryltion y AMPK, trnsfection of CGNs with constitutively ctive mutnt of AMPK (AMPKCA) resulted in decrese in phospho FOXO3 (Thr32) levels nd n increse in Bim expression (Supplementry Figures 2A nd B). Finlly, to demonstrte tht FOXO3 dephosphoryltion ws consequence of AKT downregultion, we trnsfected CGNs with constitutively

3 FOXO3 nd AMPK 3 c Shm 3 2 h 4 h 8 h 6 h 24 h Time fter glutmte tretment 5 βactin pakt(ser473) AKT β Actin Shm 3 2 h 4 h 8 h 6 h 24 h Time fter glutmte tretment Shm 3 2 h 4 h 8 h 6 h 24 h Shm 3 2 h 4 h 8 h 6 h 24 h Time fter glutmte tretment pampkα(thr72) d Time fter glutmte tretment pmtor(ser2448) mtor βactin pfoxo3(thr32) FOXO3 βactin e pakt 5 5 AMPK sirna Control sirna Shm 2 Shm 2 Time (h) fter glutmte tretment pakt (Ser473) AKT pfoxo3 (Thr32) FOXO3 βactin pfox3o3 5 5 AKTCA Control plsmid Shm 2 4 Shm 2 4 Time (h) fter glutmte tretment pfoxo3(thr32) FOXO3 AKT Figure 2 Downregultion of the mtor/akt pthwy during excitotoxicity. () CGNs exposed to GLUT/ GLY (/ mm, 3 min) incresed phospho Thr72 AMPK levels 3 min fter exposure (Po.5; n ¼ 3). ctin served s loding control. () GLUT exposure permnently decresed phospho Ser2448 mtor levels (Po.5; n ¼ 3). Totl mtor nd ctin served s loding controls. (c) GLUT reduced phospho Ser473 AKT levels min 2 h fter GLUT exposure (Po.5; n ¼ 4). Totl AKT nd ctin served s loding controls. (d) GLUT reduced phospho Thr32 FOXO3 levels 2 h fter GLUT exposure (Po.5; n ¼ 6). Totl FOXO3 nd ctin served s loding controls. (e) CGNs were trnsfected with AMPK sirna or Control sirna expressing vectors efore GLUT exposure. Phospho Ser473 AKT/ Thr32 FOXO3 levels were nlyzed ( nd 2 h) fter GLUT exposure. AMPK sirna neurons presented higher levels thn control sirna neurons t oth timepoints (Po.5; n ¼ 3). Totl AKT, FOXO3 nd ctin served s loding controls. AMPK depletion ws monitored y quntifiction of totl AMPK levels (Supplementry Figure ). (f) CGNs were trnsfected with AKTCA or control construct efore GLUT exposure. AKTCA neurons presented significntly higher phospho (Thr32) FOXO3 levels thn control neurons (2 nd 4 h) fter GLUT exposure (Po.5; n ¼ 3). Totl FOXO3 nd ctin served s loding controls. AKTCA expression ws monitored y totl AKT levels f βactin ctive mutnt of AKT (AKTCA). The expression of AKTCA prevented the decrese in phosphofoxo3 (Thr32) levels 2 nd 4 h fter GLUT exposure (Figure 2f). AMPK ctivtion during excitotoxicity induces FOXO3 nucler trnsloction. We next nlyzed whether the nucler presence of FOXO3 ws lso incresed in response to GLUT. CGNs were trnsfected with FOXO3GFP plsmid nd exposed to either GLUT/GLY (/ mm) or experimentl uffer. Confocl imges of single neurons were tken for FOXO3GFP, Hoechst nd propidium iodide (PI). Quntifiction of the GFP fluorescence intensity inside nd outside the nucleus llowed the clcultion of nucler/ cytoplsmic FOXO3GFP rtio. In cells exposed to GLUT, 29.3±8.3% of neurons showed sudden nd permnent increse in this rtio, occurring with dely tht vried etween nd 5 h fter GLUT exposure (men dely of 3 h 28±33 min) (Figures 3 nd ). Of note, ll neurons tht exhiited this sudden nd permnent increse underwent nucler shrinkge nd cell deth 7 2 h fter GLUT exposure (men timepoint 9 h 4±56 min), s indicted y the loss of the GFP fluorescence signl nd n increse in PI fluorescence. The remining neurons tht survived (62.8±2.% of the totl popultion) did not show ny chnge in their nucler/cytoplsmic rtio, or showed trnsient increse tht recovered to seline levels (men durtion of rtio increse h 2±7 min) (Supplementry Figures 3A nd B). Cells exposed to experimentl uffer only did not show ny ltertions in the nucler/cytoplsmic FOXOGFP rtio (Supplementry Figure 3C). We investigted whether AMPK ctivtion lone ws sufficient to induce FOXO3 nucler trnsloction. Expression of

4 4 FOXO3 nd AMPK 2 Nucler / Cytoplsmic FOXO3GFP rtio.5.5 FOXO3GFP Hoechst Time (h) fter glutmte tretment Time (h) fter glutmte tretment PI c FOXO3GFP distriution (% of cell popultion) Cytoplsmic Nucler CytoNucler d FOXO3GFP distriution (% of cell popultion) Cytoplsmic Nucler CytoNucler Control plsmid AKTCA e αfoxo3 distriution (% of cell popultion) Compound C Cytoplsmic Nucler CytoNucler f Nucler FOXO3 Cytoplsmic FOXO3 αfoxo3 GFP Control sirna AMPK sirna Hoechst Figure 3 AMPK ctivtion during excitotoxicity induces FOXO3 nucler trnsloction. ( d) CGNs were trnsfected with FOXO3GFP construct. () Trces of the nucler/cytoplsmic FOXO3GFP rtio in single CGNs s monitored using timelpse microscopy. Following tretment with GLUT/ GLY (/ mm) for 3 min, 29.3±8.3% of the neurons showed n increse in the nucler/cytoplsmic FOXO3GFP rtio, nd its persistence ws ssocited with drop in GFP signl nd neuronl deth s indicted y PI uptke. Representtive trces re shown. () Imges extrcted from the timelpse series showing CGN with FOXO3GFP only in the cytoplsm t the strt of the time series ( h), persistent trnsloction of FOXO3GFP to the nucleus (4.5 h), shrinkge of the nucleus (6.5 h), nd susequent secondry necrosis (2 h), r, mm. (c) FOXO3GFP ws coexpressed with the AKTCA construct or control vector. In the presence of the control vector, GLUT tretment incresed the percentge of neurons with nucler FOXO3GFP (lck rs) 3.5 h fter exposure, nd decresed the percentge with cytoplsmic FOXO3GFP (white rs) (Po.5; n ¼ 3). AKTCA expression prevented oth effects. (d) CGNs were pretreted with CC ( mm) or vehicle for 45 min efore GLUT exposure. CC pretretment significntly reduced the increse in the percentge of cells with nucler FOXO3GFP (lck rs), nd the decrese in those with cytoplsmic FOXO3GFP (white rs), 3.5 h fter GLUT exposure (Po.5; n ¼ 3). (e) CGNs were exposed to GLUT 48 h fter trnsfection with AMPK sirna or Control sirna nd were fixed nd stined 3.5 h lter with Hoechst nd n ntiody ginst FOXO3. AMPK sirna significntly reduced the GLUTinduced increse in nucler FOXO3 (lck rs) nd decrese in cytoplsmic FOXO3 (white rs), confirming the effects seen in (d)(po.5; n ¼ 4). (f) Imges of single CGNs trnsfected with GFPtgged sirna nd stined with n ntiody ginst FOXO3 (red) nd with Hoechst (lue), showing primrily nucler (imge series on left) or cytoplsmic (imge series on right) FOXO3 stining. Br, 5 mm the AMPKCA construct significntly incresed the percentge of neurons showing FOXO3GFP nucler presence when compred with cells trnsfected with control vector (Supplementry Figure 2C). We next tested whether the nucler trnsloction of FOXO3GFP during excitotoxic injury ws prevented either y expression of AKTCA, or y inhiition of AMPK. We compred the percentge of cells exhiiting nucler,

5 FOXO3 nd AMPK 5 cytoplsmic or cytoplsmic nd nucler presence of FOXO3 GFP 3.5 h fter GLUT or vehicle exposure. GLUT exposure significntly incresed the percentge of neurons with nucler FOXO3GFP compred with control neurons, nd decresed the percentge of neurons with cytoplsmic FOXO3GFP (Figures 3c nd d). Both effects were rogted y AKTCA expression (Figure 3e) or y pretretment with CC ( mm) (Figure 3d). These results were confirmed with immunofluorescence experiments on neurons trnsfected with AMPK sirna (Figures 3e nd f). These dt demonstrted tht excitotoxic injury induces FOXO3 nucler trnsloction in n AMPK nd AKTdependent mnner. FOXO3 nucler trnsloction is not sufficient for im expression nd cell deth. In ddition to FOXO3 nucler trnsloction, further posttrnsltionl modifictions my e necessry to stimulte its trnscriptionl ctivity. 7,22 This hypothesis ws tested y coexpressing the im promoter plsmid with FOXO3 construct, mutted t the AKT phosphoryltion sites nd therefore permnently loclized to the nucleus ( FOXO3nucler 2 ). GLUT/GLY (/ mm) exposure significntly incresed luciferse ctivity in CGNs expressing FOXO3nucler (Supplementry Figure 4A), suggesting tht FOXO3 required further posttrnsltionl modifictions to increse its inding to the im promoter. Additionl singlecell experiments implicted AMPK in mediting these events. Neurons trnsfected with FOXO3 GFP were exposed to GLUT, nd 2 h lter treted with CC ( mm). We hypothesized tht this tretment prdigm would void AMPK inhiition during or shortly fter GLUT exposure, llowing FOXO3 nucler trnsloction, ut inhiiting AMPK susequent to this. Indeed, lte ddition of CC did not prevent FOXO3GFP nucler trnsloction. However, ll neurons tht displyed permnent FOXO3 nucler trnsloction filed to undergo susequent nucler shrinkge nd cell deth (Figures 4 nd ), nd remined vile until termintion of the experiments. To corroorte these singlecell experiments we nlyzed Bim nd FOXO3 expression nd locliztion y western lotting in CGNs treted with CC 2 h fter GLUT exposure. CCtreted neurons expressed lower levels of Bim, without decrese in FOXO3 nucler trnsloction (Figures 4c nd d). GLUTinduced cell deth ws lso significntly impired in this setting (Figure 4e). Tken together, this dt suggest tht AMPK my hve further essentil role in im ctivtion tht extends eyond inducing FOXO3 trnsloction, nd tht requires prolonged AMPK ctivity. We lso investigted FOXO3 trnsloction nd Bim expression during vried durtions of phrmcologicllyinduced AMPK ctivity. We found tht lthough CGNs sujected to continuous AICAR tretment (2.5 mm) showed incresed Bim expression, no significnt Bim induction occurred with trnsient ddition of AICAR to the cultures for h despite FOXO3 nucler trnsloction in oth tretment prdigms. Incresed nucler locliztion of pampk ws lso oserved fter continuous, ut not trnsient, AICAR tretment (Figure 4f), confirming tht prolonged AMPK ctivtion is required for Bim expression, nd suggesting tht nucler pampk my medite this Bim expression. FOXO3 ctivtion y AMPK is coherent feedforwrd network motif tht cn ct s suppressor of trnsient stress signls. To understnd the regultory role of AMPK in FOXO3 ctivtion nd Bim expression from systems point of view, we employed computtionl modeling. Initilly, we modeled (y ordinry differentil equtions) onestep FOXO3 ctivtion where pampkmedited Akt inhiition led to FOXO3 dephosphoryltion (FOXO3 dephos ) nd ctivted FOXO3 for Bim expression (Figure 5, denoted y ()). This liner pthwy predicted similr FOXO3 ctivtion, nd therefore Bim expression, following oth shortterm nd longterm periods of AMPK ctivity (Figure 5). However, this liner model neither explined our results tht the durtion of AMPK ctivtion my determine whether Bim is expressed or not (Figure 4f), nor did it support previous findings tht shortterm pampk ctivtion ws neuroprotective.,23 We therefore ssumed tht n dditionl regultion step ws required to prevent Bim expression following shortterm AMPK ctivtion, while mintining roust Bim expression following prolonged AMPK ctivtion. We incorported into our model second ctivtion step of FOXO3 dephos y pampk ( FOXO3 mpk ) (Figure 5, dshed line denoted y (2)). In this model, Bim induction ws suppressed for shortterm pampk ctivity (Figure 5c, 4 min), s the pampk signl (second ctivtion step) ws no longer ctive y the time the signl of the first ctivtion step hd induced FOXO3 dephosphoryltion. Prolonged pampk ctivtion, however, outlsted the dely of the first step nd led to pronounced increse in Bim expression (Figure 5c, min). This gve qulittively good explntion of our findings in Figure 4f, where longterm AMPK ctivtion led to significnt Bim expression, wheres shortterm ctivtion did not. The inclusion of second FOXO3 ctivtion step y pampk resulted in network motif known s coherent feedforwrd loop (CFL). AMPK induces im expression y direct FOXO3 phosphoryltion. AMPK hs een shown to regulte the DNA inding of trnscription fctors y direct phosphoryltion.,24,25 Interestingly, in nonneuronl cells, AMPK hs een shown to specificlly ctivte FOXO3 y phosphoryltion. 24 To vlidte the modeling results we tested if AMPK phosphoryltion of FOXO3 ws required for Bim expression. We coexpressed FOXO3 construct (FOXO36A), which is mutted t the six residues (Thr79, Ser399, Ser43, Ser439, Ser555 nd Ser588) phosphorylted y AMPK, longside the im promoter reporter plsmid. As expected, CGNs cotrnsfected with FOXO3 wt nd treted with GLUT showed significnt increse in luciferse ctivity compred with control neurons. However, in neurons cotrnsfected with the FOXO36A mutnt, this effect ws rogted (Figure 6, upper pnel). These results were lso confirmed y western lot nlysis of Bim protein levels (Figure 6, lower pnel). Next we tested whether FOXO3 trnsloction during excitotoxicity lso required FOXO3 phosphoryltion y AMPK. CGNs were trnsfected with FOXO36A or FOXO3 wt constructs nd then exposed to GLUT or experimentl uffer. Three hours fter exposure cultures were sujected to

6 6 FOXO3 nd AMPK sucellulr frctiontion. GLUT tretment induced similr increse in FOXO3 wt nd FOXO36A nucler levels when compred with control neurons (Figure 6). A similr decrese ws oserved in cytosolic FOXO3 wt nd FOXO36A levels (Flg M2tg), suggesting tht FOXO3 phosphoryltion y AMPK did not ffect FOXO3 trnsloction. We lso oserved tht pampk (Thr72) levels were incresed in the nucleus nd decresed in the cytosol fter GLUT tretment (Figure 6), suggesting the nucler trnsloction of oth pampk nd FOXO3 during excitotoxicity. The Nucler / Cytoplsmic FOXO3GFP rtio Time (h) fter glutmte tretment Compound C ddition Time (h) fter glutmte tretment FOXO3GFP Hoechst PI c 5 d 5 Bim (cyto) 5 FOXO3 (cyto) 5 Bim (cyto) FOXO3 (cyto) βctin (cyto) βctin (cyto) Compound C (2 h fter Glut) FOXO3 (nucler) NeuN (nucler) Compound C (2 h fter Glut) e Cell deth (% of Pl cells) Compound C (2 h fter Glut) f Bim (cyto) Shm 2 h 4 h 8 h Time fter AICAR dministrtion (2.5 mm continuous) Shm 2 h 4 h 8 h Time fter AICAR dministrtion (2.5 mm for h) Bim (cyto) pampk (Thr72) α (cyto) FOXO3 (cyto) βctin (cyto) pampk (Thr72) α (nucler) FOXO3 (nucler) NeuN (nucler)

7 FOXO3 nd AMPK 7 fct tht nucler pampk is enriched 3 h fter GLUT exposure indictes tht FOXO3 phosphoryltion y AMPK cn occur susequent to FOXO3 nucler trnsloction. We next directly tested whether the phosphoryltion stte of FOXO3 in the nucleus depended on AMPK ctivity. CGNs trnsfected with the FOXO3nucler mutnt were pretreted with CC ( mm) nd lter exposed to GLUT. Four hours lter, we detected elevted levels of FOXO3nucler serine phosphoryltion in neurons exposed to GLUT when compred with controls. This effect ws rogted in neurons pretreted with CC (Figure 6c). The ove results showed tht FOXO3 phosphoryltion y AMPK incresed im promoter ctivtion during excitotoxic injury. This phosphoryltion ws specific to the im promoter, s FOXO36A expression did not ffect the ctivtion of chimeric promoter ering six cnonicl FOXOinding elements (TTGTTTAC ox) not present in the im promoter (Supplementry Figure 4B). We next tested if inhiition of FOXO3 phosphoryltion y AMPK could fcilitte the ctivtion of lterntive FOXO3 trgets, such s the promoter of the stress tolernce gene MnSOD. 22 A FOXO3 wt or FOXO3 6A construct were coexpressed with luciferse reporter plsmid contining the MnSOD promoter sequence (psodluc) in CGNs. GLUT exposure incresed luciferse ctivity in control neurons trnsfected with FOXO3 wt (Figure 6d). However, FOXO36A expression incresed luciferse ctivity in neurons exposed to either experimentl uffer or GLUT. This effect ws rogted y muttion of the FOXO3 inding sites in the MnSOD promoter (psodlucmut) (Figure 6d). FOXO3 ctivtion y AMPK is required for cell deth. Next, we nlyzed whether inhiition of FOXO3 phosphoryltion y AMPK decresed neuronl injury during excitotoxicity (Figure 7). We cotrnsfected CGNs with GFPexpressing plsmid nd either the FOXO3 wt, dominntnegtive FOXO3 (DNFOXO3), or FOXO36A expression plsmid. Cell deth ws determined y evluting the percentge of PI þ nd GFP þ cells efore nd 6 h fter GLUT/GLY (/ mm) exposure. As shown in Figure 7, FOXO3 wttrnsfected neurons treted with GLUT showed significntly higher levels of cell deth thn neurons exposed to vehicle. However, cell deth ws negligile in cells trnsfected with DNFOXO3 or FOXO36A. To explore the possile relevnce of FOXO3 regultion y AMPK in ischemic neuronl deth, we sujected primry mouse corticl neurons to oxygen/glucose deprivtion (OGD)induced neuronl injury. Neurons were cotrnsfected with GFP expression vector nd either DNFOXO3, or FOXO36A expression constructs. Cultures were sujected to OGD for 3 min in hypoxi chmer, nd susequently returned to normoxic/normoglycemic conditions for 24 h. Cell deth ws determined y evluting the percentge of pyknotic nuclei in the GFP þ cells. As shown in Figure 7, trnsfection with FOXO36A led to significntly lower levels of cell deth when compred with cells cotrnsfected with control plsmid, n effect tht ws comprle to the effect of DNFOXO3. Discussion The present study descries the propoptotic ctivity of the energy sensor AMPK during excitotoxic injury. Although AMPK ctivtion hs een ssocited with cell survivl,,23,26 it is now evident tht it is lso involved in longterm cell fte decisions. AMPK ctivtion hs een shown to ctivte poptosis in different cell types, 27,28 including neurons, 9,3 nd this poptosis occurs fter prolonged periods of AMPK ctivtion. 27,28 AMPK hs lso een shown to medite ischemic neuronl deth in vivo. 4 As poptosis is terminl cellulr decision, the signling pthwys controlling the propoptotic ctivities of AMPK require complex regultion. We here demonstrte tht the comined ctivtion of AP nd FOXO3 trnscription fctors y AMPK is required for the trnscriptionl ctivtion of the propoptotic im gene. Indeed, im ctivtion nd cell deth induction required oth AMPKdependent FOXO3 nucler trnsloction nd direct FOXO3 phosphoryltion y AMPK. Systems nlysis nd computtionl modeling showed tht this twostep ctivtion of FOXO3 provides mens for filtering out effects of shortterm AMPK ctivtion on im induction, while providing roust im ctivtion following prolonged AMPK ctivity. Previous work from our group hs demonstrted tht AMPK medites cell deth y expression of the propoptotic BH3 only proteins Bim nd, in some cell types, Bmf. 3,29 Bim promoter muttion of either the FOXO3 or the AP inding site prevented im ctivtion in neurons during excitotoxicity. We demonstrte tht in ddition to the enggement of multiple trnscription fctors, individul trnscription fctors my lso e suject to dditionl posttrnsltionl control steps in order to efficiently ctivte im trnscription. We oserved tht AMPK trnsiently inhiited AKT signling during excitotoxicity, nd tht AKTmedited FOXO3 dephosphoryltion ws key step tht llowed the nucler trnsloction of FOXO3. 2 This trnsloction ws permnent in neurons, which underwent poptosis, nd ws trnsient or did not occur in surviving cells. Specific mechnisms must therefore Figure 4 FOXO3 nucler trnsloction is not sufficient for im expression nd cell deth. () Trces of the FOXO3GFP rtio in single CGNs exposed to GLUT nd 2 h lter treted with CC ( mm). In this cse, ll neurons tht suffered persistent FOXO3 nucler trnsloction did not suffer susequent nucler shrinkge or cell deth until termintion of experiment. Representtive trces re shown. () Imges extrcted from the timelpse series showing CGN with FOXO3GFP in the cytoplsm t the strt of the time series ( h), persistent trnsloction of FOXO3GFP to the nucleus (6 h), no shrinkge of the nucleus (2 h), nd no susequent secondry necrosis (6 h), r, mm. (c) CC ( mm) ddition 2 h fter GLUT exposure significntly reduced the GLUTinduced increse in Bim expression (mesured 8 h fter GLUT exposure, Po.5; n ¼ 3). ctin served s loding control. (d) CC ( mm) ddition 2 h fter GLUT exposure did not prevent the GLUTinduced decrese in cytoplsmic (cyto) FOXO3 or increse in nucler FOXO3 levels (mesured 8 h fter GLUT exposure, Po.5; n ¼ 4). ctin nd NeuN served s loding controls. (e) Addition of CC ( mm) 2 h fter GLUT tretment significntly reduced the % of PI þ cells detected 6 h fter the excitotoxic insult (Po.5; n ¼ 3). (f) CGNs exposed to continuous AICAR (2.5 mm) tretment showed n upregultion of Bim levels in the cytosol, nd pampk (Thr72) nd FOXO3 levels in the nucleus (Po.5; n ¼ 3). CGNs exposed to trnsient AICAR tretment (2.5 mm, h, then wshout) lso showed incresed nucler FOXO3 levels; however, Bim levels in the cytosol nd pampk in the nucleus were not upregulted (n ¼ 3). ctin nd NeuN served s loding controls. Times (h) indicte durtion of AICAR tretment (continuous) or recovery times following wshout of AICAR

8 8 FOXO3 nd AMPK Bim Concentrtion (AU) Bim Concentrtion (AU) c mx. Model Incorporting Coherent FeedForwrd Loop (CFL) Twostep FOXO3 ctivtion mx. FOXO3 mpk Bim 2 FOXO3dephos pampk mtor AKT Shortterm pampk ctivity (4 min) Longterm pampk ctivity ( min) ' 3' 2h 4h 8h 6h 24h Simultion Time Shortterm pampk ctivity (4 min) Longterm pampk ctivity ( min) ' 3' 2h 4h 8h 6h 24h Simultion Time exist to restore pakt/pfoxo3 levels in surviving neurons. Survivl responses during excitotoxicity hve een ssocited with enhnced neuronl glucose uptke nd ATP vilility, s well s with growth fctor signling, 9,2 FOXO3kt Liner Pthwy Model [ only] Predicted Bim Expression Model Incorporting CFL [ 2 ] Predicted Bim Expression Figure 5 FOXO3 ctivtion y AMPK is coherent feedforwrd network motif tht cn ct s suppressor of trnsient stress signls. A second ctivtion step of FOXO3 is necessry to explin neuronl tolernce to shortterm pampk ctivity while mintining Bim expression following prolonged pampk ctivity. () pampkmedited FOXO3 ctivtion ws initilly modeled y liner pthwy (denoted y ()). () This liner pthwy predicted similr Bim expression ptterns following short nd longterm periods of pampk ctivity nd conflicted with recent findings of the neuroprotective role of pampk. (c) Incorportion of the second ctivtion step (denoted y (2) in ()) reduced Bim expression following shortterm pampk ctivity (4 min, gry rs). This network structure represents CFL nd, y preventing Bim expression in times of shortterm stress, my llow pampk to exert its prosurvivl effects. Significnt Bim expression is still present following longterm pampk ctivity ( min, lck rs). Brs indicte reltive differences in expression levels etween oth scenrios suggesting tht these fctors could restore pakt/pfoxo3 levels. The persistent mtor dephosphoryltion fter GLUT exposure suggests the involvement in this process of other AKT regultors esides mtor. Given the centrl role of AKT in severl cell survivl pthwys such s PI3K nd GSK3 signling, 3 the regultion of AKT independently from mtor nd AMPK pthwys is not surprising nd my involve phosphtses such s PP2A or PTEN. 3,32 The AKTdependent nucler trnsloction of FOXO3, however, ws not the sole determinnt of FOXO3dependent im gene expression. Both iochemicl nd singlecell imging experiments, s well s experiments using FOXO3 6A, demonstrted tht im ctivtion nd cell deth dditionlly required FOXO3 phosphoryltion y AMPK. In contrst, the trnscription of the MnSOD promoter ws stimulted y FOXO36A expression lone. This finding suggested tht direct phosphoryltion y AMPK could guide FOXO3 towrds its inding to the im promoter insted of towrds others trgets such s the MnSOD promoter. The twostep ctivtion of FOXO3 y AMPK is CFL network motif. Well estlished in systems iology, nd overrepresented in pthwys of gene regultion, CFLs re known to induce gene expression upon sustined, ut not trnsient signls. 33,34 Indeed, our identified CFL enles the filtering of trnsient AMPK impulses so tht shorter periods of AMPK ctivity do not led to Bim induction nd llow pampk to exert its prosurvivl effects. This filtering is chieved y the dely required for the AKTmedited FOXO3 dephosphoryltion in the first ctivtion step. The period of AMPK ctivity must outlst this dely to ctivte FOXO3 in the second ctivtion step. Incresed survivl signling, through AKT ctivtion, would prolong the dely of the first ctivtion step, requiring longer period of AMPK ctivity to induce Bim expression. This ide is supported y evidence showing reduction in poptosis following stimultion of AKT. 35 Consequently, the CFL of FOXO3 ctivtion my regulte Bim expression sed on oth stress durtion nd the extent of prosurvivl signling. Previously, we demonstrted the involvement of JNK in Bim expression nd the nucler trnsloction of CJUN induced y AMPK during excitotoxicity. 3 Neurons hve high levels of constitutive JNK ctivity, 36 so it remins to e shown whether ny surplus ctivtion of JNK y AMPK contriutes to im gene induction during excitotoxic poptosis. Such potentil third ctivtion step y AMPK cnnot e excluded, nd my integrte other stressrelted signls to form more complex cscde of CFLs. Oxidtive stress, for exmple, my ctivte the JNK/AP pthwy. 36 Similrly, we cnnot exclude role of other trnscription fctors, such s My, 8 in im ctivtion. Additionlly, the FOXO fmily of trnscription fctors my e suject to other posttrnsltionl modifictions such s JNK phosphoryltion or SIRT decetyltion. 22,37 AMPK hs previously een shown to control the DNAinding of different trnscription fctors or cofctors y phosphoryltion.,24,25 AMPK phosphoryltion of FOXO3 ws not required for its nucler trnsloction (see lso Greer et l. 24 ). However, it is possile tht FOXO3 dephosphoryltion nd its susequent nucler trnsloction is requirement for susequent posttrnsltionl modifictions (tht is, FOXO3 phosphoryltion y AMPK). 7 Indeed, this is

9 FOXO3 nd AMPK 9 Luciferse reporter ctivity (Fold increse) FOXO3 wild type FOXO3 6A Empty vector FOXO3 wild type FOXO3 6A Bim βctin 3 2 FOXO3 wild type FOXO3 6A Nucler frction Cytosolic frction Flg M2tg pampk(thr 72)α AMPKα NeuN Flg M2tg pampk(thr 72)α AMPKα βctin c FOXO3 nucler Compound C phosphoserine FlgHAtg Luciferse reporter ctivity (Fold increse) psodluc psodlucmut FOXO3 wild type FOXO3 6A Figure 6 AMPK induces im expression y direct FOXO3 phosphoryltion. () Upper. CGNs were cotrnsfected with vector contining the im promoter nd either the wt FOXO3 or FOXO36A constructs. GLUT/GLY (/ mm, 3 min) exposure significntly incresed im promoter ctivtion in cells trnsfected with FOXO3 wt (Po.5; n ¼ 4). FOXO36A expression prevented this effect (Po.5; n ¼ 4). () Lower. Western lot nlysis confirmed the effect of FOXO36A on Bim protein levels fter GLUT exposure (3 min). Results re representtive of two independent experiments. () Upper. Nucler frction of CGNs trnsfected with FOXO3Flg wt or FOXO36AFlg constructs. Flgtg detection y western lot nd densitometry nlysis showed similr upregultion of oth constructs 4 h fter GLUT exposure (Po.5; n ¼ 4). Nucler phospho (Thr72) AMPK levels were lso upregulted. NeuN ws used s nucler mrker nd ctin s loding control. () Lower. GLUT exposure downregulted levels of FOXO3 wt nd FOXO36A. Cytoplsmic phospho (Thr72) AMPK levels were lso downregulted. ctin served s loding control. Representtive lots re shown. (c) CGNs trnsfected with FOXO3nucler mutnt (tgged with flgha) nd immunoprecipted showed n increse in Serphosphoryltion 4 h fter GLUT exposure (Po.5; n ¼ 3). Pretretment with CC ( mm) for 45 min rogted this effect (Po.5; n ¼ 3). (d) CGNs were cotrnsfected with luciferse reporter contining MnSOD promoter sequence (psodluc) nd either wt FOXO3 or FOXO36A. GLUT exposure incresed luciferse ctivity in cells trnsfected with FOXO3 wt. However, FOXO36A expression incresed luciferse ctivity in neurons exposed to experimentl uffer or GLUT. This effect ws rogted y muttion of the FOXO3 inding sites in the MnSOD promoter (psodlucmut) (Po.5; n ¼ 4) d necessry condition for the functioning of the proposed CFL. We provided evidence of nucler effects of AMPK on FOXO3 with Bim induction only detected fter sustined AMPK ctivtion nd nucler presence of pampk. Shorter periods of AMPK ctivity did not led to AMPK trnsloction or Bim expression, lthough FOXO3 nucler trnsloction ws mintined in oth scenrios. This evidence suggested tht interction of FOXO3 nd AMPK in the nucleus could e requirement for im promoter ctivtion nd poptosis. Interestingly, neuronl poptosis ws recently descried in model of Huntington s disese s dependent on nucler ccumultion of AMPK. 38 In conclusion, our dt suggest tht FOXO3 ctivtion represents key step during immedited excitotoxic neuronl deth. The complex interply etween cellulr ioenergetics, AMPK ctivtion, nd mtor/akt/foxo3 signling provides moleculr frmework for cell fte decision mking, preventing unwnted poptosis ctivtion during physiologicl AMPK ctivtion or during conditions of mild ioenergetic stress.

10 FOXO3 nd AMPK Mterils nd Methods Mterils. Fetl clf serum nd miniml essentil medium were otined from Invitrogen (Bio Sciences, Dulin, Irelnd). GLUT, GLY, Hoechst nd PI were from SigmAldrich (Arklow, Irelnd). AICAR ws from Cell Signling (Isis Ltd, Bry, Irelnd). CC ws otined from Cliochem (Merck Biosciences, Nottinghm, UK). Preprtion of primry CGNs. Both mouse (C57BL) or rt (Sprgue Dwley) cereell were isolted from postntl dy 7 pups. CGN cultures were otined s descried previously. 7 Cells were plted on polylysinecoted glss Cell deth (% of ded GFP cells) FOXO3 wild type DNFOXO3 FOXO3 6A OGD / Control plsmid OGD / AMPK sirna coverslips, glss Willco dishes, 6well pltes, nd 24well pltes t 6 cells/ml, nd mintined t 37 C in humidified tmosphere of 5% CO 2 / 95% ir. Experiments were performed fter 8 dys in vitro. All niml work ws performed with ethics pprovl from the RCSI nd under licenses grnted to the uthors y the Irish Deprtment of Helth nd Children. Plsmids, sirna nd trnsfection. The FOXO3GFP plsmid in n EGFPN vector ckone ws kindly provided y MP Smidt (University Medicl Center, Utrecht, The Netherlnds). The AMPKCA construct is the truncted version of AMPK fter residue 32 nd includes Myc tg nd ws kind gift from Dvid Crling (Imperil College London, London, UK). The AKTCA construct expresses the myristoylted AKT sequence nd ws kindly provided y S Pons (Biomedicine Institute, CSIC, Brcelon, Spin). The Firefly luciferse reporter plsmid contining.8 kb of the im promoter sequence long with similr constructs with mutted FOXO3 or AP inding sites were kindly provided y Eric Lm (Imperil College London, London, UK). The pecefoxo3tm (termed here FOXO3nucler ), construct mutted t the three sites (T32A/S253A/S35A) of AKT phosphoryltion nd permnently locted in the nucleus, ws kindly provided y ME Greenerg (Hrvrd Medicl School, Boston, MA, USA). The pece FOXO3M2flg nd pecefoxo36am2flg (sextuple mutnt T79A/S339A/ S43A/S555A/S588A/S626A t the sites for AMPK phosphoryltion) were kind gifts from A Brunet (Hrvrd Medicl School). The pmxgfp construct ws otined from LONZA (Bsel, Switzerlnd). The prltk (TK Renill luciferse), the 6 DBE FOXO3 (reporter luciferse plsmid with six copies of the FOXO fmily proteininding element) nd the reporter plsmids contining the MnSOD promoter sequence (psodluc) long with similr construct with mutted FOXO3 inding sites (psodlucmut) were kind gift from BM Burgering (University Medicl Center, Utrecht, The Netherlnds). The DNFOXO3 expresses the DNAinding domin (mino cids 4 268) of pecefoxo3tm, nd ws kindly provided y I TorresAlemn (Cjl Institute, CSIC, Mdrid, Spin). sirna trgeting AMPK/2 (pfivampksirna) nd the control sequence (pfivcontrolsirna) were previously descried. CGNs were trnsfected using clcium phosphte s previously descried, the trnsfection regent Neurofect from Genlntis (AMS Biotechnology, Milton, UK), or n electroportion kit (LONZA) s per mnufcturer s instructions. OGD / DNFOXO3 OGD / FOXO3 6A GFP Signl Live cell microscopy. CGNs on glss Willco dishes (WillCo Wells BV, Amsterdm, The Netherlnds) were trnsfected with FOXO3GFP construct. After 48 h cells were treted for 3 min with GLUT nd GLY ( mm nd mm) in experimentl uffer (2 mm NCl, 3.5 mm KCl,.4 mm KH 2 PO 4, 2mM HEPES, 5 mm NHCO 3,.2 mm N 2 SO 4,.2 mm CCl 2,.2 mm MgCl 2 nd 5 mm glucose; ph 7.4). Control CGNs were treted with experimentl uffer only. Next, CGNs were stined with Hoechst nd PI t finl concentrtion of. mg/ml. Following tretment, the preconditioned medium, supplemented with HEPES (finl concentrtion 2 mm), ws replced onto the cells nd the dish ws plced on the stge of confocl microscope equipped with 63.4 NA oil immersion ojective nd thermostticlly regulted chmer t 37 C (LSM 7, Crl Zeiss, Jen, Germny). A thin lyer of minerl oil (.5 ml) ws dded onto the medium to prevent evportion. GFP ws excited t 488 nm with n Argon lser % Pyknotic Nuclei OGD Control plsmid AMPK sirna DNFOXO3 FOXO3 6A Figure 7 FOXO3 ctivtion y AMPK is required for cell deth. () CGNs were cotrnsfected with GFP nd either the FOXO3 wt, DNFOXO3 or FOXO36A expression plsmid. FOXO3 wttrnsfected neurons treted with GLUT showed significntly higher percentge of cell deth (3.4±3.8% of totl cells) thn neurons exposed to vehicle (6.4±2.7%) 6 h fter GLUT/GLY (/ mm, 3 min) exposure. However, cell deth fter the excitotoxic insult ws negligile in cells trnsfected with DNFOXO3 or FOXO36A, which presented significnt decrese in the percentge of cell deth compred with FOXO3 wttrnsfected neurons (Po.5; n ¼ 3). () Corticl neurons were cotrnsfected with GFPexpression vector nd either AMPK sirna, DNFOXO3 or FOXO36A expression constructs. After 72 h trnsfection cultures were sujected to OGD (3 min) in hypoxi chmer, nd returned to normoxic/normoglycemic conditions. Cell deth ws determined 24 h lter y evluting the percentge of pyknotic nuclei in the GFP þ cells following Hoechst stining. Expression of either FOXO36A or DNFOXO3 significntly reduced the percentge of neuronl cell deth (3.±7.8% nd 2.±7.8% of totl cells, respectively) compred with cells cotrnsfected with control plsmid (3.6±4.6%) (Po.5; n ¼ 4). Br, mm

11 FOXO3 nd AMPK (AOTF set to.5%) nd the emission collected in the rnge of nm. Hoechst ws excited t 45 nm (% trnsmission, ND filter nd AOTF) nd the emission detected in the rnge of 4 to 48 nm. For the detection of plsm memrne permeiliztion PI ws excited t 543 nm (AOTF.5% trnsmission) nd fluorescence emission ws detected ove 57 nm. Imges were cptured t 5 min intervls t.5 mm opticl slice thickness. The resulting imges were processed using MetMorph 7.5 softwre (Moleculr Devices, Wokinghm, UK). The GFP verge intensity fluorescence signl ws quntified t the nucleus nd cytoplsm to determine the nucler/cytoplsmic FOXO3GFP rtio. At lest 33 singlecell experiments were nlyzed for cells treted with GLUT, nd 6 for those treted with experimentl uffer. For quntifiction of FOXO3 nucler trnsloction in neuronl popultions, CGNs were plted on 24well pltes. At 3.5 h fter GLUT/ experimentl uffer exposure, neurons were incuted for 3 min with Hoechst (finl concentrtion mg/ml) nd PI (finl concentrtion.2 mg/ml). For ech group, 8 9 cells were nlyzed in three independent experiments using n Eclipse TE 2s inverted microscope (Nikon, Amstelveen, The Netherlnds) nd 4.6 NA ojective. Imges of GFP, PI nd Hoechst signls of single neurons were tken nd processed with Imge J softwre (NIH, Bethesd, MD, USA) to determine the nucler/cytoplsmic FOXO3GFP rtio. Neurons were scored s follows: FOXO3GFP cytoplsmic (rtio o.9), FOXO3GFP nucler (rtio 4.), FOXO3GFP cytoplsmic/nucler (.9ortioo.). Western lotting nd immunoprecipittion. Western lotting ws performed s descried. Neurons were lysed with PIK uffer (% NP4, 5 mm NCl, 2 mm Tris, ph 7.4, % glycerol, mm CCl 2, mm MgCl 2, 4 mm sodium vndte,.2 mm PMSF, mg/ml leupeptin, mg/ml protinin,.% phosphtse inhiitor cocktils I nd II of SigmAldrich). Blots were proed with rit polyclonl ntiodies to Bim (H9) nd HA (Y) from Snt Cruz Biotechnology (Dulin, Irelnd). Phospho (Thr72) AMPK ntiody, totl AMPK, phospho (Thr83/Thr85) JNK, totl JNK, phospho (Ser473) AKT, totl AKT, phospho (Ser63) cjun, phospho (Ser2448) mtor totl mtor, phospho (Ser253) FOXO3 nd phospho (Thr32) FOXO3 ll from Cell Signling nd ntimyc tg nd totl FOXO3 ntiodies from Millipore (Crrigtwohill, Irelnd). Blots were lso proed with mouse monoclonl ntiodies to ctin (clone DM A) nd Antiflg M2 from SigmAldrich, nd NeuN nd AntiPhosphoserine clone 4A4 from Millipore. Horserdish peroxidseconjugted secondry ntiodies from Thermo Scientific (Fisher, Dulin, Irelnd) were detected using SuperSignl West Pico Chemiluminescent Sustrte (Thermo Scientific) nd imged using FujiFilm LAS3 imging system (FujiFilm, Dulin, Irelnd). To normlize for protein lod, memrnes were relotted (ReBlot, Millipore) nd incuted with n pproprite control ntiody. Levels of the protein under study were expressed reltive to protein lod in ech lne s determined y pproprite control protein content. pampk levels were normlized to ctin, s GLUT tretment decresed totl AMPK levels. Densitometry nlysis ws performed using Imge J softwre (NIH). A representtive lot is shown from totl of t lest three independent experiments, except where indicted. Immunoprecipittion ws performed in cultured neurons lysed in PIK uffer nd centrifuged t 22 g for 2 min, nd superntnts were incuted with ntiodies ound to 25% grose (Snt Cruz Biotechnology) overnight. The immunoprecipittes were wshed three times with the sme lysis uffer, resuspended in 2.5 SDS loding uffer nd nlyzed y western lot. Cytosolic nd nucler frctiontion. Nucler nd cytosolic frctions were otined from totl neuronl lystes s descried. 39 The qulity of the frctiontion ws determined y ssying for the presence of the nucler protein NeuN. Cell deth ssys. CGNs were cultured on 24well pltes. For determintion of poptotic morphology cells were stined live with Hoechst t finl concentrtion of mg/ml. After incution for min, nucler morphology ws oserved nd cells with condensed nuclei were scored s pyknotic nd expressed s percentge of the totl popultion. For determintion of neuronl mortlity cells were trnsfected with pmxgfp nd different constructs under evlution in : 5 rtio. GFPpositive cells (GFP þ cells) were counted efore tretment to determine the seline level of trnsfected cells t timepoint, nd were counted gin 2 h fter tretment. Results were expressed s percentge of GFP þ cells t time. Neurons were counted in 2 different fields using n Eclipse TE 3 inverted microscope (Nikon) nd 2.45 NA ojective. All ssys were done in triplicte in t lest three independent experiments. Luciferse ssys. CGNs were trnsfected with luciferse reporter constructs contining different promoters long with Renill luciferse expression plsmid (prltk) for normliztion. Neurons were lysed in pssive lysis uffer nd luciferse ctivity ws nlyzed using luminometer nd dul luciferse ssy kit ccording to the mnufcturer Promeg (Medicl Supply, Dulin, Irelnd). Trnsfections were performed in triplicte dishes nd reltive luciferse counts were normlized using TK Renill luciferse cotrnsfection. Bckground luminescence ws sutrcted, nd Luciferse ctivity ws expressed s fold of the increse with respect to the control. Immunocytochemistry. Following tretment, CGNs cultured on glss coverslips were fixed (3% prformldehyde), permeilised (.% Triton x in PBS), locked (5% horse serum (Invitrogen Bio Sciences),.3% Triton x in PBS) nd incuted with n ntiody ginst FOXO3 (Millipore), t room temperture for 2 h. FOXO3 stining ws detected with n ntirit Alex Fluor568conjugted secondry ntiody (Invitrogen Bio Sciences). Nuclei were lso stined with Hoechst CGNs were imged on the LSM 7 microscope detiled ove nd excited using 56, 488 nd 45 nm lsers. Imges were nlyzed using ImgeJ. The cytoplsmic nd nucler FOXO3 verge intensity ws quntified nd CGNs were scored s cytoplsmic, nucler or cyto/nucler s descried ove. Gene trgeted mice. The genertion nd genotyping of im / mice hs een previously descried. 4 The im / mice were originlly generted on mixed C57BL/6 29SV genetic ckground, using 29SVderived ES cells, ut hd een ckcrossed for 42 genertions onto the C57BL/6 ckground. Oxygen nd glucose deprivtion in corticl neurons. Corticl neurons cultured on 24well pltes were trnsfected with pmxgfp plsmid nd different constructs under evlution in : 5 rtio. Before OGD induction, cells were rinsed in prewrmed glucosefree medi nd trnsferred to the neroic chmer. The neroic chmer hd n tmosphere consisting of.5% O 2,5% CO 2 nd 85% N 2. Cells were incuted in OGD medi consisting of 2 mm CCl 2, 25 mm NCl, 25 mm NHCO 3, 2.5 mm KCl,.25 mm NH 2 P 4, 2 mm MgSO 4 nd mm Sucrose nd hd ph of 7.4. After 3 min of OGD, cells were returned to normoxic conditions contining 2% O 2 nd 5% CO 2. Control cells remined in normoxic conditions throughout. After 24 h cells were stined live with Hoechst nd cell deth ssessed s descried ove. Computtionl modeling. We modeled the AMPKdependent FOXO3 ctivtion y trnslting the rection networks shown in Figure 5 into Ordinry Differentil Equtions. As model input, different ctivtion profiles of AMPK (pampk) were resemled y Gussin curve with mximum of 27 nm t reference timepoint nd fullwidthhlfmxim etween nd 5 min to mimic trnsient nd prolonged ctivtion. In first ctivtion step, phosphorylted AMPK (pampk) ws ssumed to inhiit mtor nd AKT ctivity, leding to dephosphoryltion of FOXO3 (FOXO3 dephos ). In second ctivtion step (Figure 5 (2)), pampk directly phosphorylted FOXO3 dephos to frction (FOXO3 AMPK ) ssumed to e cple of inducing im trnscription. The rection network is depicted in Supplementry Tle. Protein protein interctions, including ll phosphoryltion nd dephosphoryltion events, were modeled y mss ction kinetics, wheres Hill kinetic ws ssumed for im trnscription. Firstorder degrdtion kinetics were ssumed for dephosphorylted proteins. Phosphorylted protein frctions were ssumed to recover to stedystte level. The complete Mtl code is ville y request from the uthors. Sttisticl nlysis. Dt re expressed s men±s.d. Differences mong groups were nlyzed y onewy ANOVA followed y Tukey test. Comprison etween two groups ws performed with the t test. Po.5 ws considered significnt. Conflict of Interest The uthors declre no conflict of interest. Acknowledgements. This study ws supported y Mrie Curie IEF (PIEFGA ), Science Foundtion Irelnd (8/ IN/ 949), nd the Helth Reserch Bord in Irelnd (PHD/27/). We thnk Dr. Hns Georg Koenig for discussions nd support, In Woods nd Srh Cnnon for excellent