Amyloid-β interrupts the PI3K-Akt-mTOR signaling pathway

Amyloid-b Interrupts the PI3K-Akt-mTOR Signaling Pathway That Could Be Involved in Brain-Derived Neurotrophic Factor-Induced Arc Expression in Rat Cortical NeuronsTsan-Ju Chen,1*Dean-Chuan Wang,2and Shun-Sheng Chen31Department of Physiology,Faculty of Medicine,College of Medicine,Kaohsiung Medical University, Kaohsiung,Taiwan2Faculty of Sports Medicine,College of Medicine,Kaohsiung Medical University,Kaohsiung,Taiwan3Department of Neurology,Chang Gung Memorial Hospital-Kaohsiung Medical Center,Chang Gung University College of Medicine,Kaohsiung,TaiwanThe deposition of amyloid-b(A b)contributes to the pathogenesis of Alzheimer’s disease.Even at low levels, A b may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory.Brain-derived neurotrophic factor(BDNF)is well known to be capable of inducing the synthesis of activity-regulated cytoskeleton-associated protein(Arc),which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar A b at a nonlethal level was sufficient to impair BDNF-induced Arc expression in cultured rat cortical neurons.In this study,BDNF treatment alone induced the activation of the phosphatidylinositol3-kinase-Akt-mammlian target of rapamycin(PI3K-Akt-mTOR)signal-ing pathway,the phosphorylation of eukaryotic initiation factor4E binding protein(4EBP1)and p70ribosomal S6 kinase(p70S6K),the dephosphorylation of eukaryotic elongation factor2(eEF2),and the expression of Arc. Interrupting the PI3K-Akt-mTOR signaling pathway by inhibitors prevented the effects of BDNF,indicating the involvement of this pathway in BDNF-induced4EBP1 phosphorylation,p70S6K phosphorylation,eEF2dephos-phorylation,and Arc expression.Nonlethal A b pretreat-ment partially blocked these effects of BDNF.Double-immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2dephosphorylation and Arc expression following BDNF treatment regardless of the presence of A b.These results reveal that,in cul-tured rat cortical neurons,A b interrupts the PI3K-Akt-mTOR signaling pathway that could be involved in BDNF-induced Arc expression.Moreover,this study also provides thefirst evidence that there is a close correlation between BDNF-induced eEF2dephosphorylation and BDNF-induced Arc expression.V C2009Wiley-Liss,Inc.Key words:Alzheimer’s disease;eEF2;synaptic plasticity;cultured cortical neuronsOne of the pathological hallmarks of Alzheimer’s disease(AD)is extracellular accumulation of senile pla-ques composed of primarily aggregated amyloid-b(A b) peptide(Mesulam,1999).The deposition of A b,leading to neuronal loss and subsequent cognitive impairment, contributes to the pathogenesis of AD(Mesulam,1999). However,cognitive impairment may precede both high levels of A b accumulation and massive neuronal loss in the brain(Mucke et al.,2000;Hardy and Selkoe,2002). It has been suggested that A b at low levels might inter-fere with the signaling cascades critical for neuronal function and thus lead to cognitive impairment in the early stage of AD(Tong et al.,2001).Synaptic plasticity representing activity-dependent modification of synaptic strength underlies cognitive functions such as learning and memory.The hippocam-pal long-term potentiation(LTP),induced by high-frequency stimulation(HFS),is frequently used as a cellular model for synaptic plasticity.Two phases of LTP are recognized,including an early phase,dependent on modifications of existing proteins,and a late phase, requiring new synthesis of mRNAs and proteins (Kandel,2001).Acute intrahippocampal infusion of brain-derived neurotrophic factor(BDNF),a member of the neurotrophin family,also leads to LTP(BDNF-LTP),which is dependent on protein synthesis as wellContract grant sponsor:National Science Council in Taiwan;Contract grant number:NSC-94-2320-B-037-019.*Correspondence to:Tsan-Ju Chen,PhD,Department of Physiology, Faculty of Medicine,Kaohsiung Medical University,100Shih-Chuan1st Road,Kaohsiung807,Taiwan.E-mail:tsanju@.twReceived17November2008;Revised8January2009;Accepted18 January2009Published online19March2009in Wiley InterScience(www. ).DOI:10.1002/jnr.22057Journal of Neuroscience Research87:2297–2307(2009)'2009Wiley-Liss,Inc.(Kovalchuk et al.,2002;Messaoudi et al.,2002;Kan-hema et al.,2006).A unique immediate early gene product named activity-regulated cytoskeleton-associated pro-tein(Arc)is one of the proteins whose synthesis can be induced by BDNF(Yin et al.,2002;Ying et al.,2002).A role of Arc in synaptic plasticity as well as learning and memory is implied by the fact that Arc mRNA is rapidly transcribed in response to neuronal activity and then transported to activated dendritic regions,where it undergoes local translation(Link et al.,1995;Lyford et al.,1995;Bramham et al.,2008).Inhibition of Arc protein synthesis can impair the synaptic plasticity and memory consolidation(Guzowski et al.,2000;Steward and Worley,2002).Thus,BDNF-induced Arc expres-sion appears to play a fundamental role in the stabiliza-tion of synaptic plasticity(Ying et al.,2002;Bramham and Messaoudi,2005).Recent work has shown that protein synthesis underlying activity-dependent synaptic plasticity is crit-ically controlled at the level of mRNA translation,in which the initiation and elongation steps are both highly regulated(Rhoads,1999;Bradshaw et al.,2003;Klann and Dever2004;Soule´et al.,2006).Two of the key translation factors involved are eukaryotic initiation fac-tor4E(eIF4E)and eukaryotic elongation factor2 (eEF2).Phosphorylation of eIF4E at the Ser209site is correlated with enhanced rates of translation(Gingras et al.,2004),whereas phosphorylation of eEF2at the Thr56site arrests translation elongation(Browne and Proud,2002).In addition,a serine-threonine kinase, mammalian target of rapamycin(mTOR),is also known to be involved in the translation initiation and elonga-tion steps.Two main targets of mTOR are the binding protein of eIF4E(4EBP)and the p70ribosomal S6 kinase(p70S6K).mTOR-mediated phosphorylation of 4EBP facilitates the release of eIF4E and allows its par-ticipation in translation initiation.On the other hand, phosphorylated mTOR can phosphorylate p70S6K at the Thr389site,followed by phosphorylation of eEF2 kinase(eEF2K)at the Ser366site,which facilitates the dephosphorylation of eEF2and thus stimulates protein synthesis(Wang et al.,2001;Li et al.,2005).In addition, another initiation factor,eIF4B,has to be phosphoryl-ated by the p70S6K to associate with the translation ini-tiation complex that can recognize the cap structure of mRNA,leading to the initiation of translation(Swiech et al.,2008).Consistent with thesefindings,HFS-LTP and BDNF-LTP,both dependent on protein synthesis, are impaired by treatment with the mTOR inhibitor rapamycin(Tang et al.,2002;Cracco et al.,2005),indi-cating that the activation of mTOR signaling may lead to plasticity-related protein synthesis.The well-defined BDNF signaling cascades include the phosphatidylinositol-3kinase(PI3K),the extracellu-lar signal-regulated kinase(ERK),and the phospholipase C-g(PLC-g)pathways(Kaplan and Miller,2000;Soule´et al.,2006).Among them,PI3K plays an important role in the regulation of mTOR function in conditions of protein synthesis-dependent late-phase LTP(Ray-mond et al.,2002;Karpova et al.,2006)and BDNF-activated translation(Schratt et al.,2004;Jaworski et al., 2005;Kumar et al.,2005).In addition,BDNF-LTP and stable HFS-LTP require ERK activation and translation of Arc mRNA(Ying et al.2002;Kanhema et al.,2006). BDNF alone has been reported to enhance translation elongation through activating the mTOR signaling path-way followed by the down-regulation of eEF2phospho-rylation(Inamura et al.,2005).However,some studies suggest that Arc mRNA may undergo maintained or enhanced translation under conditions of enhanced eEF2 phosphorylation(Scheetz et al.,2000;Chotiner et al., 2003;Bramham et al.,2008).According to thesefind-ings,which signaling pathway is involved in BDNF-induced Arc translation requires further investigation.For AD brains,a disturbed mRNA translation (Langstrom et al.,1989;Li et al.,2005)has been reported.In neuroblastoma cells treated with A b and in transgenic mice carrying AD-related genes,an impaired mTOR-dependent pathway has also been found(Lafay-Chebassier et al.,2005).However,little attention is paid to the direct effects of A b on translation of plasticity-related proteins.Although our recent study showed the first evidence that BDNF-induced Arc expression is impaired by the treatment of A b at5l M in cultured cortical neurons(Wang et al.,2006),the underlying molecular mechanism is unclear.Therefore,further investigations were performed in the current study.The results showed that,after pretreatment of A b at5l M, BDNF-induced activation of the PI3K-Akt-mTOR sig-naling was interrupted,leading to a reduction in both BDNF-induced eEF2dephosphorylation and BDNF-induced Arc expression.MATERIALS AND METHODS ChemicalsSynthetic A b1–42peptides were obtained from Biosource (Camarillo,CA).Hank’s balanced salt solution(HBSS),fetal bovine serum(FBS),Dulbecco’s modified Eagle’s medium (DMEM),B27supplement,Glutamax-I,penicillin,and strep-tomycin were purchased from Gibco(Grand Island,NY). Nupage Bis-Tris gel,MOPS running buffer,and Nupage transfer buffer were purchased from Invitrogen(Carslbad, CA).Anti-Arc monoclonal antibody was purchased from Santa Cruz Biotechnology(Santa Cruz,CA).Antiphospho-Akt(Ser473),antiphospho-mTOR(Ser2448),antiphospho-4EBP1(Thr37/Thr46),antiphospho-p70S6K(Thr389),anti-phospho-eEF2(Thr56),anti-Akt,anti-p70S6K,and anti-eEF2 polyclonal antibodies were purchased from Cell Signaling (Beverly,MA).Antiactin monoclonal antibody and horseradish peroxidase-conjugated antibodies were obtained from Chemi-con(Temecula,CA).Alexa Fluor488(green)-conjugated anti-mouse IgG and Alexa Fluor546(red)-conjugated anti-rabbit IgG antibodies were obtained from Molecular Probes(Eugene, OR).Mounting medium was purchased from Vector Laborato-ries(Burlingame,CA).Wortmannin and rapamycin were pur-chased from Tocris Cookson Ltd.(Bristol,United Kingdom). Recombinant human BDNF,cytosine arabinoside,and other2298Chen et al.Journal of Neuroscience Researchchemicals for buffers were purchased from Sigma (St.Louis,MO).Fibrillar A b PreparationSynthetic monomeric A b 1–42with a final concentration at 100l M was prepared in PBS and incubated at 378C for 96hr to form fibrillar A b .The solution was then centrifuged at 14,000g for 10min to collect pellets and dissolve them to 100l M as a stock solution.The formation of fibrillar A b was examined by Western blot analysis.Previous study has revealed that fibrillar A b preparations typically contain >60-kDa large aggregates and include less abundant tetramer,whereas oligo-meric A b assemblies range from 30to 60kDa (Stine et al.,2003).In our preparations probed with monoclonal antibody 4G8(recognizing A b residues 17–24;Signet),unaggregated synthetic A b contained predominantly monomers (4kDa),whereas 90–130-kDa large aggregates with very few oligomers were shown after incubation for 96hr,indicating that fibrillar A b was formed (Fig.1).Cell CultureAll experiments were performed according to the guide-lines of the Institutional Animal Care and Use Committee of Kaohsiung Medical University.Primary cortical neurons were prepared from neonatal Sprague-Dawley rats as previously established (Wang et al.,2006).In brief,whole cortex cut into small pieces was digested with 0.25%trypsin/EDTA in HBSS without calcium or magnesium at 378C for 15min.The pieces were then gently rinsed in HBSS,washed twice in plating medium (10%FBS in DMEM),and gently triturated in plating medium through a fire-polished Pasteur pipette.The cell suspension was centrifuged at 800rpm for 5min.Pellets were resuspended,and then 4–53105cells/cm 2were plated on poly-D-lysine-precoated plastic dishes.Cultures were maintained at 378C in a 5%CO 2incubator.After 24hr,the medium was replaced with DMEM containing 2%B27supplement,500l M Glutamax,100U/ml penicillin,and 100l g/ml streptomycin.Cytosine arabinoside at 5l M was alsoadded to inhibit the proliferation of nonneuronal cells.All cultures were maintained for 10days in vitro,with medium changed every 3days.Western Blot AnalysisCells were lysed in ice-cold RIPA lysis buffer contain-ing inhibitors of proteinase and of phosphatase (50mM HEPES,pH 7.5,150mM sodium chloride,1%deoxycholate,1%NP-40,0.1%sodium dodecyl sulfate,1mM EGTA,5mM EDTA,1mM dithiothreitol,1mM sodium fluoride,2mM sodium orthovanadate,1mM phenylmethylsulfonyl fluoride,10l g/ml aprotinin,10l g/ml leuteptin)to avoid degradation and dephosphorylation of proteins.Samples were then spun down at 14,000g at 48C for 20min.The superna-tant was quantified for total protein concentration using the Pierce Bradford Protein Assay Kit.Protein samples (30or 50l g per sample)were resolved by 10%Bis-Tris gel using MOPS running buffer for 30min under 200V,120mA volt-age and current setting.Proteins were then transferred to a polyvinylidene difluoride (PVDF)membrane using NuPage transfer buffer for 90min under 30V,170mA voltage and current setting.Membranes were incubated at room tempera-ture in TTBS (0.1%Tween 20,0.05M Tris in 0.9%NaCl,pH 7.4)containing 5%nonfat milk for 60min to block the nonspecific binding.After incubation with the primary anti-bodies that recognize Arc (1:400),phospho-Akt (1:1,000),phospho-mTOR (1:1,000),phospho-4EBP1(1:1,000),phos-pho-p70S6K (1:1,000),or phospho-eEF2(1:1,000)for 2hr at room temperature or at 48C overnight,the blots were washed in TTBS and then incubated with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:2,000)for 1.5hr.Blots were then washed three times with TTBS.Immuno-labeling was detected using enhanced chemiluminescence reagents (Santa Cruz Biotechnology),and the protein levels were quantified by densitometric analysis.The immunoblots with phosphorylation site-specific antibodies were subse-quently stripped with 62.5mM Tris-HCl,pH 6.8,containing 2%SDS,and 100mM 2-mercaptoethanol for 30min at 508C and were reprobed with antibodies against actin (1:10,000),Akt (1:1,000),mTOR (1:1,000),p-70S6K (1:1,000),or eEF2(1:1,000).The detection reaction was started from the block-ing step as described above.ImmunocytochemistryCultured neurons were fixed with 4%paraformaldehyde in PBS for 10min at room temperature and were washed with PBS.Then,cells were incubated overnight at 48C in PBS containing 5%normal goat serum and 0.1%Triton X-100to block nonspecific binding.Thereafter,cells were incu-bated in PBS containing anti-Arc monoclonal antibody (1:100)and antiphospho-eEF2polyclonal antibody (1:500)for 24hr at 48C.Alexa Fluor 488(green)-conjugated anti-mouse IgG and Alexa 546(red)-conjugated anti-rabbit IgG were used as secondary antibodies.Finally,cells were mounted with Antifade Mounting Media containing 4,6-diamino-2-pheno-lindol dihydrochloride (DAPI)to reveal the nuclei.All images were taken under an Axioscope fluorescent microscope (Zeiss,Oberkochen,Germany)with the same exposure timeandFig.1.Formation of fibrillar A b 1–42was confirmed by Western blot analysis.Synthetic monomeric A b 1–42was incubated in PBS at 378C for 96hr to form fibrillar A b .The molecular weight of unaggregated monomeric A b 1–42was 4kDa (lane A).Fibrillar A b 1–42consisted primarily of 90–130-kDa large aggregates (lane B).Asterisk indicates A b oligomers.A Mechanism of A b -Reduced Arc Expression 2299Journal of Neuroscience Researchwere analyzed in Image Pro-Plus software(Media Cybernet-ics).The numbers of Arc-and phospho-eEF2-immunoreac-tive neurons were counted through a320objective and visualized through a camera connected to a microscope on a computer screen on which a10310square grid was applied. Immunopositive neurons were summed for eachfield and di-vided by the total number of nuclei to give the percentages of Arc-or phospho-eEF2-immunoreactive cells in thatfield. Statistical AnalysisResults of densitometric analysis from Western blot were expressed as the mean6SEM of three or more inde-pendent experiments.Quantification of immunoreactivities is represented as the percentage of immunoreactive neurons, expressed as the mean6SD.Statistical significance was ana-lyzed by one-way ANOVA and post hoc test.The relation-ship between Arc and phospho-eEF2immunoreactivities was analyzed by the bivariate Pearson correlation analysis.The sig-nificance level was set at P<0.05.RESULTSBoth PI3K and ERK Signaling Pathways Participate in BDNF-Induced Arc Expression Because both PI3K and ERK signaling pathways are essential to protein synthesis-dependent LTP,the roles of PI3K and ERK in BDNF-induced Arc expression were examined.Neonatal rat cortical neurons cultured for10 days in vitro were pretreated with the PI3K inhibitor wortmannin(1l M)or the MEK inhibitor PD98059(100 l M)for10min,followed by an incubation with BDNF (50ng/ml)for60min.Wortmannin or PD98059signifi-cantly reduced the levels of BDNF-induced phospho-Akt or phospho-ERK,respectively,indicating that the inhibi-tors used here were specific and effective(data not shown).As for the Arc expression,the basal level was very low,but an obvious enhancement of Arc expression was seen after treating cultures with BDNF.However,the pretreatments with wortmannin or PD98059significantly reduced the levels of BDNF-induced Arc expression(Fig. 2A).These results suggest that both PI3K and ERK sig-naling pathways are involved in BDNF-induced Arc expression in neonatal rat cortical neurons.PI3K but Not ERK Signaling Pathway Contributes Predominantly to BDNF-Induced Changes in the Phosphorylation States of4EBP1and eEF24EBP and eEF2are two major downstream effectors of the mTOR signaling.Their phosphorylation states are correlated with the translation initiation and elongation steps,respectively.Therefore,the effects of inhibiting PI3K or ERK signaling pathways on BDNF-induced changes in the phosphorylation states of4EBP1and eEF2 were examined.Cultured cortical neurons were treated as described previously.As shown in Figure2B,C,only wortmannin pretreatment could prevent BDNF-induced 4EBP1phosphorylation and eEF2dephosphorylation. The pretreatment with PD98059did not alter the effects of BDNF on4EBP1and eEF2.These results indicated that the PI3K signaling pathway played a predominant role in the effects of BDNF on4EBP1and eEF2.There-fore,the role of the PI3K-Akt-mTOR signaling pathway was further evaluated in the following experiments.BDNF Induced Both eEF2Dephosphorylation and Arc Expression in a Time-Dependent Manner The phosphorylation state of eEF2plays a crucial role in the elongation step.Although some studies have suggested that Arc translation may occur under condi-tions of enhanced eEF2phosphorylation,our results shown in Figure2A,C reveal that BDNF can enhance Arc expression and reduce eEF2phosphorylation.To evaluate whether the effect of BDNF on thephospho-Fig.2.Effects of interrupting the PI3K or ERK signaling pathways on the expression of Arc,phospho-4EBP1,or phospho-eEF2after BDNF treatment.Neonatal rat cortical neurons cultured for10days were treated with BDNF alone(B,50ng/ml)for60min or pre-treated with PI3K inhibitor wortmannin(W,1l M)or MEK inhibi-tor PD98059(P,100l M)for10min,followed by incubated with BDNF for60min.After BDNF treatments,the levels of Arc and p-4EBP1were significantly increased,whereas the level of p-eEF2 was significantly decreased.Pretreatment of wortmannin prevented these effects of BDNF,whereas pretreatment of PD98059prevented only BDNF-induced Arc expression.For quantitative densitometry, band intensities were normalized to actin for the corresponding lane. Data for p-4EBP1(B)and p-eEF2(C)are shown as mean6SEM with control group normalized to100%.However,the basal level of Arc(A)was very low,so data are shown as mean6SEM with BDNF group normalized to100%.Statistical significance was ana-lyzed by one-way ANOVA and a post hoc test.*P<0.05,**P< 0.01compared with the control group.#P<0.05,##P<0.01com-pared with the BDNF group.2300Chen et al.Journal of Neuroscience Researchrylation state of eEF2is correlated with the expression of Arc induced by BDNF,neonatal rat cortical neurons cultured for 10days in vitro were treated with BDNF (50ng/ml)for various periods (0–60min).As shown in Figure 3,the levels of phosphorylated eEF2(p-eEF2)decreased gradually with time while the expression of Arc increased robustly by 60min,indicating that BDNF induced both eEF2dephosphorylation and Arc expres-sion in a time-dependent manner.Blocking PI3K-Akt Signaling PreventsBDNF-Induced Activation of the mTOR-eEF2Pathway and BDNF-Induced Arc Expression SimultaneouslyWith the results shown in Figure 2,the role of the PI3K-Akt-mTOR signaling pathway in BDNF-induced Arc expression was evaluated.Neonatal rat cortical neu-rons cultured for 10days in vitro were pretreated with the PI3K inhibitor wortmannin (1l M)for 10min,fol-lowed by an incubation with or without BDNF (50ng/ml)for 60min,and then the levels of phospho-Akt (p-Akt),Akt,phospho-mTOR (p-mTOR),mTOR,phospho-eEF2(p-eEF2),eEF2,and Arc were measured by Western blot analysis.As shown in Figure 4,the basal levels were low for p-Akt,p-mTOR,and Arc but high for p-eEF2.BDNF treatments enhanced the expressions of p-Akt,p-mTOR,and Arc but reduced the expression of p-eEF2simultaneously.Pretreatment of wortmannin prevented these effects of BDNF.After treating cultures with wortmannin alone,the expressions of various phos-phorylated proteins and Arc were almost the same as thebasal levels.In addition,the levels of total proteins including Akt,mTOR,and eEF2did not change signifi-cantly following various treatments,indicating that the phosphorylation state instead of the levels of these pro-teins was influenced by BDNF and/or wortmannin.As mentioned above,4EBP and p70S6K are two main targets of mTOR.Phosphorylated 4EBP partici-pates in the step of translation initiation,and phosphoryl-ated p70S6K contributes to the translation in both the initiation and the elongation steps.Thus,a further experiment was used to evaluate the changes in the phosphorylation states of 4EBP1and p70S6K.As shown in Figure 5A,the levels of p-4EBP1and p-p70S6K were increased after BDNF treatments.Pretreatment with wortmannin prevented these effects of BDNF.Wort-Fig. 3.BDNF induced both eEF2dephosphorylation and Arc expression in a time-dependent manner.Neonatal rat cortical neu-rons cultured for 10days in vitro were treated with BDNF (50ng/ml)for the indicated time (0–60min).After BDNF treatment,eEF2phosphorylation (p-eEF2)decreased gradually with time,while the expression of Arc increased robustly by 60min.For quantitative den-sitometry,band intensities were normalized to actin for the corre-sponding lane.Data for p-eEF2are shown as mean 6SEM with control group normalized to 100%.Statistical analysis was performed as for Figure2.Fig. 4.Interruption of the PI3K-Akt signaling prevents BDNF-induced activation of the mTOR-eEF2pathway and BDNF-induced Arc expression.Neonatal rat cortical neurons cultured for 10days were treated with the PI3K inhibitor wortmannin (W,1l M)for 10min,followed by incubated with or without BDNF (B,50ng/ml)for 60min.After BDNF treatments,the levels of p-Akt,p-mTOR,and Arc were significantly increased,whereas the level of p-eEF2was significantly decreased.Pretreatment with wortmannin prevented these effects of BDNF,but no effect was found after treating cultures with wortmannin alone.The levels of Akt,mTOR,and eEF2did not change significantly with various treatments.For quantitative densitometry,band intensities for p-Akt,p-mTOR,p-eEF2,and Arc were normalized to Akt,mTOR,eEF2,and actin,respectively,for the corresponding lane.Data for p-Akt (A ),p-mTOR (B ),and p-eEF2(C )are shown as mean 6SEM with control group normal-ized to 100%.Because the basal level of Arc (D )was very low,data are shown as mean 6SEM with BDNF group normalized to 100%.Statistical analysis was performed as for Figure 2.A Mechanism of A b -Reduced Arc Expression 2301Journal of Neuroscience Researchmannin alone did not change the basal levels of p-4EBP1or p-p70S6K.Blocking mTOR-Dependent Signaling Prevents BDNF-Induced eEF2Dephosphorylation and BDNF-Induced Arc Expression SimultaneouslySubsequently,the role of mTOR in BDNF-induced Arc expression was evaluated.Cortical neurons cultured for 10days in vitro were pretreated with the mTOR in-hibitor rapamycin (1l M)for 10min,followed by an incubation with or without BDNF (50ng/ml)for 60min.In Figure 6,pretreatment with rapamycin prevented the effects of BDNF on the levels of p-mTOR,p-eEF2,and Arc but not on p-Akt levels.Treating cultures with rapamycin alone had no effects on the levels of p-Akt,p-mTOR,p-eEF2,and Arc compared with their controls.In addition,no change was found in the levels of total proteins including Akt,mTOR,and eEF2following vari-ous treatments,indicating that the phosphorylation state instead of the levels of these proteins was influenced by BDNF and/or rapamycin.In addition,similarly to the results of wortmannin pretreatment,rapamycin also reduced BDNF-enhanced phosphorylation of 4EBP1and p70S6K,whereas rapamycin alone did not change the basal levels of 4EBP1or p70S6K (Fig.5B).Pretreatment With A b Prevents the Effects of BDNF on the PI3K-Akt-mTOR Pathway,the Phosphorylation State of eEF2,and the Expression of ArcBased on the results of MTT viability assay,our recent study showed that the treatment with fibrillar A b 1–42at 5l M for 6hr was nontoxic to cultured corti-cal neurons but was sufficient to impair BDNF-induced Arc expression (Wang et al.,2006).Further experiments were performed in the current study to examine whether the PI3K-Akt-mTOR signaling pathway was involved in A b -impaired BDNF-induced Arc expres-sion.Cultured cortical neurons were pretreated with 5l M of fibrillar A b 1–42for 6hr,followed by an incu-bation with or without BDNF (50ng/ml)for 60min.In Figure 7,pretreatment with A b 1–42reduced the enhanced effects of BDNF on the levels of p-Akt,p-mTOR,and Arc and prevented BDNF-induced eEF2dephosphorylation.After treatment of cultures with A b 1–42alone,the levels of p-Akt,p-mTOR,p-eEF2,and Arc were similar to their basal levels.No change was found in the levels of total proteins including Akt,mTOR,and eEF2following various treatments,indicat-ing that the phosphorylation state instead of the levels of these proteins was influenced by BDNF and/or A b 1–42.As for the phosphorylation states of 4EBP1and p70S6K,pretreatment with A b 1–42reduced the enhanced effects of BDNF,as shown in Figure 5C.Furthermore,double-immunofluorescent staining was carried out with antibodies against p-eEF2and Arc to reveal the relationship between eEF2phosphorylation and Arc expression after various treatments.In the non-treated control group,cortical neurons exhibited strong fluorescence for p-eEF2in both cell body and dendrites but showed weak staining for Arc (Fig.8A–C).After the treatment with BDNF,an opposite result was observed;neurons showed strong immunoreactivities for Arc in both cell body and dendrites but presented much lower immunoreactivities for p-eEF2(Fig.8D,E).OnlyFig.5.Pretreatments with wortmannin (A ),rapamycin (B ),or A b 1–42(C )reduced the enhanced effects of BDNF on the phosphorylation of 4EBP1and p70S6K.Neonatal rat cortical neurons cultured for 10days were treated with the PI3K inhibitor wortmannin (W,1l M)or the mTOR inhibitor rapamycin (R,1l M)for 10min or with5l M A b 1–42for 6hr,followed by incubation with or without BDNF (B,50ng/ml)for 60min.Representative blots showed that all pretreatments reduced BDNF-enhanced phosphorylation of 4EBP1and p70S6K.2302Chen et al.Journal of Neuroscience Research。