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Transient Phosphatidylinositol 3-Kinase Inhibition Protects Immature Primary Cortical Neurons from Oxidative Toxicity via Suppression of Extracellular Signal-regulated Kinase Activation*

  • David J. Levinthal
    Affiliations
    Center for Neuroscience, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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  • Donald B. DeFranco
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology, University of Pittsburgh School of Medicine, E1352 BST, Pittsburgh, PA 15261. Tel.: 412-624-4259; Fax: 412-648-1945;
    Affiliations
    Center for Neuroscience, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

    Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant R01 NS38319 (to D. B. D.), National Institute of Health Predoctoral Institutional NRSA Grant T32 NS07433 (to D. J. L.), and National Institutes of Health Predoctoral Individual NRSA Grant F30 NS43824 (to D. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:January 08, 2004DOI:https://doi.org/10.1074/jbc.M314261200
      Oxidative stress has been shown to underlie a diverse range of neuropathological conditions. Glutamate-induced oxidative toxicity is a well described model of oxidative stress-induced neurodegeneration that relies upon the ability of extracellular glutamate to inhibit a glutamate/cystine antiporter, which results in a depletion of intracellular cysteine and the blockade of continued glutathione synthesis. Glutathione depletion leads to a gradual toxic accumulation of reactive oxygen species. We have previously determined that glutamate-induced oxidative toxicity is accompanied by a robust increase in activation of the mitogen-activated protein kinase (MAPK) member extracellular-signal regulated kinase (ERK) and that this activation is essential for neuronal cell death. This study demonstrates that delayed ERK activation is dependent upon the activity of phosphoinositol-3 kinase (PI3K) and that transient but not sustained PI3K inhibition leads to significant protection of neurons from oxidative stress-induced neurodegeneration. Furthermore, we show that transient PI3K inhibition prevents the delayed activation of MEK-1, a direct activator of ERK, during oxidative stress. Thus, this study is the first to demonstrate a novel level of cross-talk between the PI3K and ERK pathways in cultured immature cortical neuronal cultures that contributes to the unfolding of a cell death program. The PI3K pathway, therefore, may serve opposing roles during the progression of oxidative stress in neurons, acting at distinct kinetic phases to either promote or limit a slowly developing program of cell death.
      Neuronal cell death due to oxidative stress links such diverse conditions as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and stroke (
      • Coyle J.T.
      • Puttfarcken P.
      ,
      • Mattson M.P.
      • Duan W.
      • Pedersen W.A.
      • Culmsee C.
      ). Glutamate-induced oxidative toxicity has become an excellent paradigm for studying the effects of oxidative stress in primary immature neuronal culture (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ,
      • Ratan R.R.
      • Murphy T.H.
      • Baraban J.M.
      ,
      • Li Y.
      • Maher P.
      • Schubert D.
      ,
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). In this model, inhibition of a glutamate/cystine antiporter deprives cells of essential precursors for glutathione synthesis. An increased load of ROS
      The abbreviations used are: ROS, reactive oxygen species; PBS, phosphate-buffered saline; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; DIV, days in vitro; Ara-C, cytosine-arabinoside; ANOVA, analysis of variance.
      1The abbreviations used are: ROS, reactive oxygen species; PBS, phosphate-buffered saline; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; DIV, days in vitro; Ara-C, cytosine-arabinoside; ANOVA, analysis of variance.
      results from this glutathione depletion (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ,
      • Li Y.
      • Maher P.
      • Schubert D.
      ) and activates intracellular signaling events that engage an apoptotic-like cell death program (
      • Ratan R.R.
      • Murphy T.H.
      • Baraban J.M.
      ).
      The MAPK family member ERK is activated by a vast array of stimuli that impinge upon the cell and acts on a diverse range of cellular targets (
      • Davis R.J.
      ,
      • Garrington T.P.
      • Johnson G.L.
      ,
      • Grewal S.S.
      • York R.D.
      • Stork P.J.
      ). In neurons, ERK can function to either support cell survival or promote cell death. ERK was originally implicated in neuronal cell survival in differentiated PC12 cells that required neurotrophic factor support (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ), and many subsequent studies have confirmed its contribution to neuronal cell survival in other systems (
      • Bonni A.
      • Brunet A.
      • West A.E.
      • Datta S.R.
      • Takasu M.A.
      • Greenberg M.E.
      ,
      • Gonzalez-Zulueta M.
      • Feldman A.B.
      • Klesse L.J.
      • Kalb R.G.
      • Dillman J.F.
      • Parada L.F.
      • Dawson T.M.
      • Dawson V.L.
      ,
      • Han B.H.
      • Holtzman D.M.
      ,
      • Zhu Y.
      • Yang G.Y.
      • Ahlemeyer B.
      • Pang L.
      • Che X.M.
      • Culmsee C.
      • Klumpp S.
      • Krieglstein J.
      ). However, in many other models of neuronal cell death, ERK activation has been found to be associated with cell death (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ,
      • Murray B.
      • Alessandrini A.
      • Cole A.J.
      • Yee A.G.
      • Furshpan E.J.
      ,
      • Alessandrini A.
      • Namura S.
      • Moskowitz M.A.
      • Bonventre J.V.
      ,
      • Namura S.
      • Iihara K.
      • Takami S.
      • Nagata I.
      • Kikuchi H.
      • Matsushita K.
      • Moskowitz M.A.
      • Bonventre J.V.
      • Alessandrini A.
      ,
      • Du S.
      • McLaughlin B.
      • Pal S.
      • Aizenman E.
      ,
      • Mori T.
      • Wang X.
      • Jung J.C.
      • Sumii T.
      • Singhal A.B.
      • Fini M.E.
      • Dixon C.E.
      • Alessandrini A.
      • Lo E.H.
      ,
      • Noshita N.
      • Sugawara T.
      • Hayashi T.
      • Lewen A.
      • Omar G.
      • Chan P.H.
      ,
      • Stanciu M.
      • DeFranco D.B.
      ). The mechanisms that underlie such diametric effects of ERK are unclear but could be based on differences in both the temporal and spatial pattern of ERK activation induced by the various treatments (
      • Stanciu M.
      • DeFranco D.B.
      ).
      The PI3K-Akt pathway has been found to consistently serve a pro-survival function in neurons exposed to various apoptosis-inducing stimuli (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). These effects are thought to occur via Akt-mediated inactivation of Bad (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ), caspase-9 (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), or members of the Forkhead transcription factor family (
      • Bonni A.
      • Brunet A.
      • West A.E.
      • Datta S.R.
      • Takasu M.A.
      • Greenberg M.E.
      ) among others. In neurons, there is much evidence to support significant cross-talk between PI3K and ERK (
      • Perkinton M.S.
      • Sihra T.S.
      • Williams R.J.
      ,
      • Perkinton M.S.
      • Ip J.K.
      • Wood G.L.
      • Crossthwaite A.J.
      • Williams R.J.
      ,
      • York R.D.
      • Molliver D.C.
      • Grewal S.S.
      • Stenberg P.E.
      • McCleskey E.W.
      • Stork P.J.
      ,
      • Lin C.H.
      • Yeh S.H.
      • Lu K.T.
      • Leu T.H.
      • Chang W.C.
      • Gean P.W.
      ,
      • Crossthwaite A.J.
      • Hasan S.
      • Williams R.J.
      ) with the potential for PI3K to act as a required upstream activator of ERK.
      We have previously demonstrated in immature primary cortical neurons that activation of ERK is necessary for glutamate-induced oxidative toxicity (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). We now demonstrate that this ERK activation is PI3K-dependent and that transient but not sustained PI3K inhibition leads to significant protection of neurons. Thus, the PI3K pathway may serve opposing roles during the progression of oxidative stress in neurons, acting at distinct kinetic phases to either promote or limit a slowly developing program of cell death.

      EXPERIMENTAL PROCEDURES

      Primary Cortical Cultures—Cortices from embryonic day 17 Sprague-Dawley rat fetuses (Hilltop Lab Animals, Scottdale, PA) were dissected and manually dissociated by repeated trituration using fire-polished glass pipettes in Hanks' balanced salt solution (5.4 mm KCl, 0.3 mm Na2HPO4, 0.4 mm KH2PO4, 4.2 mm NaHCO3, 137 mm NaCl, 5.6 mm d-glucose, pH 7.4) without Ca2+ or Mg2+ (Invitrogen) followed by passage through a 40-μm cell strainer (BD Biosciences) to remove clumped cells. Cells were counted and plated on 50 μg/ml poly-d-lysine-treated culture plates at a density of ∼2.1 × 104 cells/cm2. Cell viability was routinely greater than 80% as assessed by uptake of trypan blue dye upon plating. Cultures were maintained for 3–4 days in medium (DMEM (Invitrogen), 10% fetal calf serum (Hyclone, Logan, UT), 10% Ham's F-12 nutrient supplement (Invitrogen), 1.9 mm glutamine, 24 mm Hepes, and 4.5 mg/ml glucose) at 37°C and 5% CO2. All of the experiments were performed on DIV 3–4-day-old cultures to avoid the confounding effect of functional ionotropic glutamate receptor expression, which is not present in immature cultures but which begins soon after this time period (
      • Mizuta I.
      • Katayama M.
      • Watanabe M.
      • Mishina M.
      • Ishii K.
      ). Unless otherwise stated, all of the chemicals and reagents used were purchased from Sigma.
      Cell Line Culture—HT22 cells, a hippocampal cell line that is particularly sensitive to glutamate-induced oxidative toxicity (
      • Davis J.B.
      • Maher P.
      ), were maintained in DMEM supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), 100 units of penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2.
      Cell Viability—18 h following the initiation of all of the treatments, cultures were incubated for 10 min with 2 μl (1:1000 dilution) of a 6.25 mg/ml solution of propidium iodide (PI) to visualize dead or dying PI-positive cells. Cells were observed under an inverted fluorescence microscope equipped with phase-contrast optics (Nikon Eclipse TE200), and PI-labeled and unlabeled cells were counted. The percentage of labeled cells in each field was then calculated (∼200 cells/field at ×400). Three random fields were counted for each condition in at least three separate cultures.
      Western Blot Analysis—Cells were treated as described, scraped and collected into phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4), pelleted at 2–3 × 103 rpm for 5 min, and lysed in lysis buffer (50 mm Tris-Cl, pH 7.5, 2 mm EDTA, 100 mm NaCl, 1% Nonidet P-40, 100 μm NaVO4, 100 μm NaF, 2 mm dithiothreitol) supplemented with 5 μl of protease inhibitor mixture/ml of lysis buffer. Total protein concentrations were determined using the Bio-Rad kit. Equivalent amounts of total protein (either 20 or 30 μg) were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% dry milk in PBS, 0.1% v/v Tween 20. Membranes were incubated with primary antibodies (anti-phospho-ERK, anti-total ERK, anti-phospho-Akt, and anti-total-Akt, all from Cell Signaling, Beverly, MA) overnight at 4 °C with 3% dry milk or 5% bovine serum albumin, washed 3 × 10 min with PBS, 0.1% v/v Tween 20, and then exposed to the appropriate horseradish-peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were again washed 3 × 10 min with PBS, 0.1% v/v Tween 20, and immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham Biosciences) using standard x-ray film (Eastman Kodak Co.). Several different exposure times were used for each blot to ensure linearity of band intensities.
      MEK Kinase Activity Assay—MEK kinase activity was determined via the ability of immunoprecipitated MEK to phosphorylate purified unphosphorylated glutathione S-transferase-ERK2 protein using 32P-labeled ATP. 400 μg of total lysate protein from primary immature cortical cultures were pre-cleared with 50 μl of a 100 mg/ml stock of protein A-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C on a rotating shaker. The supernatants were then immunocomplexed with a nonspecific rabbit IgG antibody (fluorescein isothiocyanate-conjugated rabbit anti-sheep) or a rabbit polyclonal antibody directed against the N-terminal region of MEK-1 (anti-MEK-1 NT, Upstate Biotechnology, Lake Placid, NY) overnight at 4 °C on a rotating shaker. The immunocomplexes were absorbed to 80 μl of the 100 mg/ml stock of protein A-Sepharose beads for 2 h at 4 °C on a rotating shaker and subsequently washed twice with ice-cold lysis buffer (see above). The pelleted beads were resuspended in 50 μl of kinase buffer (lysis buffer + 50 mm MgCl2 and 100 μm ATP) supplemented with 10 μCi of 32P-labeled ATP (Amersham Biosciences) and 0.250 μg of non-phosphorylated glutathione S-transferase-ERK2 (Upstate Biotechnology) per reaction. The kinase reaction was maintained at 37 °C for 25 min and terminated by pelleting the Sepharose beads and adding 10 μlof6× Laemmli buffer to the individual supernatant fractions, which were then placed on ice. After boiling for 10 min, 20 μl of each of the samples was loaded onto a 10% polyacrylamide gel and electrophoresed at 150 V for 1 h. Autoradiographic exposures of the gels were performed for 4–12 h at –80 °C.
      Indirect Immunofluorescence—Cortical cells were plated directly onto 0.05 mg/ml poly-d-lysine coated 12-well tissue culture plates. Cells were treated with or without 5 μm cytosine-arabinoside (Ara-C) at DIV 2. On DIV 4, cells were washed twice with PBS, fixed in 4% paraformaldehyde for 18 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked for 1 h with PBS supplemented with 10% goat serum (Invitrogen) at room temperature. The fixed cells were incubated with anti-GFAP (1:500) in PBS with 10% goat serum overnight at 4 °C and washed three times with PBS before incubation with a fluorescent-labeled secondary antibody (1:1000 anti-rabbit IgG conjugated to Alexa Fluor 488, Molecular Probes, Eugene, OR) and the nuclear stain 4′,6-diamidino-2-phenylindole (1:1000). Images were viewed with a Zeiss Axiophot inverted fluorescence microscope, and the number of glial cells was counted in three random fields and calculated as a percentage of total cells.
      Detection of Intracellular ROS—Intracellular ROS was measured as described previously (
      • Hsieh H.
      • Cheng C.
      • Wu S.
      • Chiu J.
      • Wung B.
      • Wang D.
      ). Primary cortical cells were treated as described, and the dye DCFH-DA (50 μm) (Molecular Probes) was added for 1 h to establish a stable intracellular level of the probe. DCFH-DA is taken up by cells where it is converted by esterases to DCFH, and is in turn oxidized to DCF in the presence of ROS. The extent of DCF fluorescence is therefore a direct readout of intracellular oxidative stress. After 1 h, cells were washed with Hanks' balanced salt solution, scraped from their plates, and measured for DCF fluorescence intensity (excitation 475, emission 525) in a fluorometer (Wallac Victor2, PerkinElmer Life Sciences). Cell counts from each sample were used to normalize the DCF signal intensity. A 500 μm dose of H2O2 (2 h) was used as a positive control for the detection of oxidative stress. Data were expressed as the fold-change in DCF signal intensity compared with control cells within each independent experiment.
      Statistics—One-way ANOVA was performed with Bonferroni's post-hoc correction for multiple comparisons. p values <0.05 were taken to be significant.

      RESULTS

      LY294002 Protects Primary Mixed Cortical Cultures from Glutamate-induced Oxidative Toxicity—Primary immature cortical neuronal cultures (DIV 3–4) underwent an apoptotic cell death within 24 h of exposure to 5 mm glutamate (
      • Ratan R.R.
      • Murphy T.H.
      • Baraban J.M.
      ). This glutamate-induced oxidative toxicity has been extensively used as a model for oxidative stress in neurons (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ,
      • Ratan R.R.
      • Murphy T.H.
      • Baraban J.M.
      ,
      • Li Y.
      • Maher P.
      • Schubert D.
      ,
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). We have previously determined that glutamate-induced oxidative toxicity can be abrogated in cortical cultures by administration of the MEK inhibitor U0126 (10 μm) (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). Mixed cultures of immature cortical cells (DIV 3) were either left untreated, treated with 5 mm glutamate, or treated with 5 mm glutamate administered with 10 μm U0126 for 24 h. As shown in Fig. 1, A and B, U0126 protects these cells from glutamate-induced oxidative toxicity. To examine the role of PI3K-Akt in glutamate-induced oxidative toxicity, we used various doses of the specific PI3K inhibitor, LY294002. Because the PI3K-Akt signaling pathway has been so reliably associated with neuroprotection in a wide variety of neurotoxicity models, we anticipated that the administration of LY294002 would not be able to prevent glutamate-induced oxidative toxicity. Surprisingly, LY294002 conferred significant neuroprotection against glutamate-induced oxidative toxicity at doses ranging from 10 to 40 μm (Fig. 1B, 20 and 40 μm doses shown).
      Figure thumbnail gr1
      Fig. 1Representative phase-contrast images (×400 magnification) demonstrating glutamate-induced oxidative toxicity in primary cortical neurons and neuroprotection via PI3K or MEK-1 inhibition. A, immature primary cortical cultures (DIV 3) were either left untreated or treated with 5 mm glutamate for 24 h. B, cultures were treated with glutamate plus 20 μm LY294002, 40 μm LY294002, or 10 μm U0126. Both compounds led to significant neuroprotection from glutamate-induced oxidative toxicity as assessed 24 h after the initiation of glutamate treatment.
      To quantify the ability of these inhibitors to abrogate glutamate-induced oxidative toxicity, we employed the PI-uptake assay, which utilizes the property of this fluorescent dye to be taken up selectively by dying or dead cells but not by viable cells. PI-labeled and unlabeled cells were counted in three random fields per experimental condition in at least three separate cultures 18 h after the initial exposure to glutamate. At this time point, cells destined to die have begun to retract their processes, to have shrunken cell bodies, and to display condensed nuclei. The vast majority of cells treated with glutamate displayed all of these features within 20–24 h and became detached from the plate soon after this time. Fig. 2 demonstrates the dose dependence for the neuroprotective effects of LY294002. Doses of LY294002 ranging from 1 to 5 μm did not abrogate glutamate-induced oxidative toxicity, whereas higher doses (e.g. 10–30 μm) significantly protected cells (p < 0.001). The highest dose of LY294002 (e.g. 40 μm) had a slightly less protective effect, which approached significance. The administration of 20 μm alone did not lead to cell death (Fig. 4C), but 40 μm LY294002 alone exhibited some toxicity (Fig. 2A). Wortmannin, which inhibits PI3K through a different mechanism than LY294002 but is much less stable in aqueous solution (
      • Walker E.H.
      • Pacold M.E.
      • Perisic O.
      • Stephens L.
      • Hawkins P.T.
      • Wymann M.P.
      • Williams R.L.
      ), also protected against glutamate-induced oxidative toxicity and was not toxic when administered alone (Fig. 2C).
      Figure thumbnail gr2
      Fig. 2The neuroprotective effect of LY294002 is dose-dependent, and concurrent inhibition of both MEK and PI3K can potentiate neuroprotection from glutamate-induced oxidative toxicity. A, primary immature cortical cultures (DIV 3) were treated with 5 mm glutamate and increasing single doses of LY294002 (1–40 μm), two doses (20–30 μm) of LY294002 each administered 6 h apart (i.e. one with the initial treatment of glutamate and another 6 h later), 10 μm U0126, or 40 μm LY294002 alone. 18 h after initial treatment, cells were incubated for 10 min with PI to visualize dead or dying PI-positive cells using an inverted fluorescence microscope equipped with phase-contrast optics. Both PI-positive and PI-negative cells were counted, and the percentage of PI-positive cells was calculated for three random fields in at least three separate cultures. LY294002 exhibited significant protection at doses of 10 μm or higher while giving two separate doses of LY294002 abrogated its protective effect. B, subthreshold doses of U0126 can potentiate the protective effect of LY294002. A subthreshold dose of U0126 (0.5 μm) alone or with increasing doses of LY294002 was administered at the initiation of 5 mm glutamate treatment. Although 0.5 μm U0126 did not significantly protect cultures from glutamate toxicity, the lowest significantly neuroprotective dose of LY294002 was 2-fold lower (i.e. 5 versus 10 μm) in the presence of 0.5 μm U0126. There was a very strong trend for neuroprotective effects, even with the 1 μm LY294002 dose in the presence of 0.5 μm U0126. C, wortmannin protects primary cortical cultures from oxidative toxicity. Either a 100 or 200 nm dose of wortmannin was given alone or concurrently with glutamate, and toxicity was measured using PI. Both doses significantly protect neurons from cell death, and neither is toxic alone. Interestingly, a 6-h delay in the administration of LY294002 (30 μm) abolishes its ability to protect neurons from oxidative toxicity. *, p < 0.05 and ***, p < 0.001; U, U0126; LY, LY294002; W, wortmannin.
      Figure thumbnail gr4
      Fig. 4LY294002 is metabolized or sequestered by glial cells in mixed cortical cultures. Western blots were performed using whole cell lysate protein (20 μg/lane) from neuron-enriched cultures and HT22 cells (a hippocampal cell line). Blots were probed with anti-phospho-Akt-specific antibodies, stripped, and reprobed with anti-total Akt antibodies. A, the transient inhibition of PI3K (as assessed by phosphorylated Akt) observed in mixed cortical cultures is not seen in neuron-enriched cultures. Neuron-enriched cultures were obtained by treating mixed cultures (DIV 2) with 5 μm cytosine-β-d-Ara-C for 2 days. These cultures were then treated with a 20 μm dose of LY294002 for either 1 or 10 h, and whole cell lysate protein was collected for Western blot analysis. B, LY294002-induced PI3K inhibition is prolonged in HT22 cells. HT22 cells were treated with 10 μm LY294002 for 1, 2, 4, 6, or 9 h, and whole cell lysates were collected for Western blot analysis. C, the toxicity of various doses of LY294002 in mixed- and neuron-enriched cortical cultures was assessed by PI uptake (see ) 18 h after treatment. Neither 20 μm LY294002 administered in one single dose or in two repeated doses 6 h apart were toxic to mixed-cortical cultures. However, single doses of LY294002 (10–30 μm) were significantly toxic to neuron-enriched cultures. Ara-C represent neuron-enriched cultures. ***, p < 0.001; LY, LY294002.
      To examine whether a subthreshold 0.5 μm U0126 dose (i.e. a dose that did not by itself protect neurons from glutamate-induced oxidative toxicity; Fig. 2B) could potentiate the neuro-protective effect of LY294002, 0.5 μm U0126 and increasing doses of LY294002 (1–40 μm) were co-administered to cortical cultures with 5 mm glutamate. In the presence of this subthreshold dose of U0126, the minimum dose of LY294002 that was found to be significantly neuroprotective was 5 μm, a 2-fold lower dose than observed in the absence of U0126 (Fig. 2B). However, the 1 μm LY294002 dose approached significance (p < 0.012 before the stringent Bonferroni post-hoc correction for multiple comparisons) in its neuroprotective effect. These results suggest that some cross-talk between the PI3K-Akt and MEK-ERK pathways may be operating in these neurons undergoing oxidative stress.
      The manner in which LY294002 was administered was critical for its neuroprotective effect. For example, one bolus of inhibitor (40 μm) given at the initiation of glutamate treatment led to some protection from toxicity (p < 0.034 before post-hoc correction, Fig. 2A). However, administering one 20 μm dose of LY294002 at the initiation of glutamate treatment and a subsequent 20 μm dose 6 h later completely abolished the neuroprotective effect of the drug (Fig. 2A). This phenomenon was also observed for an analagous 30–30 μm LY294002-dosing schedule (Fig. 2A). The administration of two 20 μm doses of LY294002 6 h apart by itself was not toxic to the cells (Fig. 4C). Furthermore, delaying the administration of LY294002 by 6 h after the addition of glutamate abolished any protective effect of the compound (Fig. 2C). This evidence would suggest that some critical PI3K-dependent event occurs within 6 h, but not later, that is necessary for glutamate-induced oxidative toxicity.
      Both LY294002 and Wortmannin Only Transiently Inhibit PI3K in Mixed Cortical Cultures—To confirm that LY294002 and wortmannin were indeed inhibiting the PI3K-Akt pathway in our cultures, we administered the drugs for various amounts of time and monitored levels of phosphorylated Akt as an output measure of PI3K activity. As shown in Fig. 3A, in primary immature mixed cortical cultures, a single LY294002 treatment (20 μm) only transiently inhibited Akt phosphorylation. This dose had conferred significant neuroprotection from glutamate-induced oxidative toxicity (Figs. 1B and 2A). This was similarly observed in wortmannin-treated cultures (Fig. 3B). Consistently, phosphorylated Akt returned to base-line levels within ∼6 h after the initial administration of both drugs. Thus, LY294002 and wortmannin administration to mixed primary cortical cultures both inhibit PI3K transiently and have no long term effect (i.e. beyond 6 h) on the PI3K-Akt pathway.
      Figure thumbnail gr3
      Fig. 3Both LY294002 and wortmannin only transiently down-regulate phosphorylated Akt in primary immature cortical cultures. Representative Western blot (n = 3) performed using whole cell lysate protein (20 μg/lane) from cultures treated for 1, 2, 3, 6, or 10 h with LY294002 (20 μm)(A), wortmannin (100 nm)(B), or from untreated control cultures is shown. Blots were probed with anti-phospho-Akt-specific antibodies, stripped, and reprobed with anti-total Akt antibodies. LY, LY294002; W, wortmannin.
      LY294002 Is Sequestered or Metabolized by Glial Cells in Mixed Cortical Cultures—Because LY294002 is known to be stable in aqueous solution, the apparent loss after 6 h of its efficacy to inhibit PI3K action on one of its downstream targets (i.e. Akt) would be consistent with metabolism or sequestration of the compound. To further explore this phenomenon of transient PI3K inhibition in our culture system, we asked whether the presence of glial elements in the system was responsible for this apparent LY294002 inactivation. Our cultures typically contained ∼20–25% GFAP-positive cells at DIV 3–4 as determined by immunocytochemical staining (20.3% ± 7.2 S.E.). The anti-mitotic agent Ara-C was added to the mixed cultures, and LY294002 was administered 2 days later after the vast majority of glial cells were eliminated. The efficacy of Ara-C in eliminating GFAP-positive cells was verified by immunocytochemistry (i.e. fewer than 1% of cells were GFAP-positive). As shown in Fig. 4A, a single administration of 20 μm LY294002 to these enriched neuronal cultures was very effective in inhibiting PI3K activity for a prolonged period of time (i.e. 10 h). This implies that the transient inhibition of PI3K activity in mixed cultures given a single dose of LY294002 may be due to glial metabolism or sequestration of the drug. To further test this hypothesis, we administered a lower dose of LY294002 (10 μm) to the hippocampal cell line, HT22. These cultures are devoid of glial cells, and therefore, we expected LY294002 to have a prolonged effect in inhibiting PI3K. Indeed, as shown in Fig. 4B, a single dose of LY294002 (10 μm) was sufficient to inhibit PI3K for extended periods of time. Furthermore, low doses of LY294002 were toxic to HT22 cells (data not shown), which confirms that prolonged maintenance of PI3K activity plays an important role in neuronal cell survival.
      To further confirm that the maintenance of PI3K activity is important to the survival of primary cortical neurons, we measured the viability of cells in Ara-C-treated neuron-enriched cultures following treatment with LY294002. As shown in Fig. 4C, 20 μm LY294002, administered in one single dose or two doses 6 h apart to mixed cortical cultures, shows no toxic effect. However, in neuron-enriched cultures, single doses of LY294002 ranging from 10 to 30 μm showed significant neurotoxicity (Fig. 4C, p < 0.001). Thus, prolonged PI3K inhibition alone is sufficient to undermine cell survival in our cultures.
      Transient Early PI3K Inhibition Prevents the Development of a Delayed ERK Activation—We have previously determined that glutamate-induced oxidative toxicity is associated with a delayed chronic activation of ERK1/2 and that this activation is necessary for neuronal cell death (Figs. 1 and 2A) (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). In cortical cultures, we typically observe this delayed activation at 10 h or more after the initiation of glutamate treatment, which correlates with the times of increasing oxidative stress in these cells (Fig. 7) (
      • Li Y.
      • Maher P.
      • Schubert D.
      ). Therefore, we asked whether LY294002 or wortmannin, which both protect these cultures from glutamate-induced oxidative toxicity (Figs. 1 and 2), interfere with the development of this delayed ERK1/2 activation. As shown in Fig. 5A, 30 μm LY294002, a dose that significantly protected cortical neurons, is effective in preventing the delayed activation of ERK1/2. Furthermore, treatment with either 100 or 200 nm wortmannin showed attenuation of the ERK activation measured 10 h after glutamate administration (Fig. 5B). Interestingly, a 6-h delay in the administration of LY294002 after glutamate, a protocol that failed to protect cortical neurons from oxidative toxicity (Fig. 2C), abolished the ability of the compound to prevent the later ERK activation seen during oxidative toxicity (data not shown). Thus, only an early transient inhibition of PI3K activity in immature cortical neurons can inhibit the delayed activation of ERK that accompanies glutamate-induced oxidative stress.
      Figure thumbnail gr7
      Fig. 7Neither PI3K nor MEK inhibition prevents the development of glutamate-induced oxidative stress in immature primary cortical cultures. Cells were treated with glutamate for 4 or 10 h or 10 h with either LY294002 (30 μm) or U0126 (10 μm), 2 h with 500 μm H2O2, or were left untreated. 1 h prior to analysis, cells were loaded with 50 μm DCFH-DA. Cells were washed in Hanks' balanced salt solution, collected, and tested for the intensity of DCF fluorescence (excitation 475, emission 525). DCF intensities were adjusted to cell counts, and the signal was compared with untreated cells (fold-change). *, p < 0.05; ***, p < 0.001
      Figure thumbnail gr5
      Fig. 5PI3K inhibition prevents the delayed activation of ERK in glutamate-treated immature primary cortical cultures. A, 30 μm LY294002 prevents the delayed activation of ERK because of 5 mm glutamate exposure. Whole cell lysates from cultures treated with glutamate and with or without 30 μm LY294002 for varying amounts of time were compared with control lysates. ERK activation is typically observed at 10 h or later in this system. B, both 100 and 200 nm doses of wortmannin attenuate ERK activation at 10 h. Blots were probed with anti-phospho-ERK antibodies, stripped, and reprobed with anti-total ERK antibodies (20 μg of total protein were loaded/lane). A representative blot (n = 4) is shown. LY, LY294002; U, U0126; W, wortmannin.
      PI3K Inhibition Prevents the Delayed Activation of MEK-1— PI3K has been found to exert either a stimulatory or inhibitory effect on ERK activation depending, in part, upon the identity and strength of the applied extracellular stimulus (
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Wennstrom S.
      • Downward J.
      ,
      • Moelling K.
      • Schad K.
      • Bosse M.
      • Zimmermann S.
      • Schweneker M.
      ). In cases where PI3K is necessary for ERK activation, B-Raf appears to be a major conversion point for these two pathways, especially in neurons (
      • York R.D.
      • Molliver D.C.
      • Grewal S.S.
      • Stenberg P.E.
      • McCleskey E.W.
      • Stork P.J.
      ). Because MEK-1 is the major target of all Raf isoforms, we used an immunoprecipitation kinase assay to monitor changes in MEK-1 activity in extracts prepared from primary neurons treated with glutamate in the presence or absence of LY294002. In this way, we could determine whether PI3K was acting at the level of ERK or on upstream kinases such as Raf/MEK-1 during the development of the PI3K-dependent delayed ERK activation. As is shown in Fig. 6, glutamate-induced oxidative toxicity in primary immature cortical cells leads to the delayed recruitment of active MEK-1 at 10 h, which is consistent with the observed onset of ERK activation. This result is not surprising given that the MEK-1 inhibitor U0126 was previously shown to prevent the development of ERK activation at this time point (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ). However, the administration of LY294002 at the initiation of glutamate treatment prevented the recruitment of MEK-1 activity at 10 h, showing that the late recruitment of MEK-1 is dependent upon early but not late PI3K activity (Fig. 6). Thus, PI3K activity must affect the ERK pathway at the level of MEK-1 or upstream of MEK-1 during glutamate-induced oxidative toxicity, formally eliminating the possibility that PI3K activity influences ERK phosphorylation independent of MEK-1.
      Figure thumbnail gr6
      Fig. 6PI3K inhibition prevents the delayed recruitment of MEK-1 activity in glutamate-treated immature primary cortical cultures. MEK-1 immunocomplexes were isolated using protein A-Sepharose beads, and MEK-1 activity was determined via the incorporation of 32P into purified unphosphorylated glutathione S-transferase-ERK2 protein (see “Experimental Procedures”). MEK-1 activity was strongly induced at 10 h, and early LY294002 administration abrogated this induction. *, negative control (control lysates were immunocomplexed with a nonspecific fluorescein isothiocyanate-conjugated rabbit anti-sheep antibody); **, negative control (the 10-h glutamate sample was processed as specified with the exception that 10 μm U0126 was added into the second lysis buffer wash and during the kinase reaction. LY, LY294002; U, U0126.
      Neither PI3K Inhibition nor MEK Inhibition Prevents Oxidative Stress in Primary Mixed Cortical Cultures—To test the hypothesis that the protective effects of PI3K inhibition or MEK inhibition may be attributed to the prevention of oxidative stress in our cultures, we measured ROS production using the dye DCF. As can be seen in Fig. 7, a significant increase in DCF fluorescence intensity is seen at 10 h but not at 4 h after the administration of glutamate. A high dose of H2O2 was used as a positive control in the assay. Interestingly, neither LY294002 (30 μm) nor U0126 (10 μm) significantly effected the extent of oxidative stress measured at 10 h after glutamate administration. This finding implies that the loss of ERK activation at 10 h (via PI3K inhibition or direct MEK inhibition) can uncouple cells from oxidative toxicity despite the ongoing presence of oxidative stress.

      DISCUSSION

      The PI3K-Akt pathway is well recognized for its ability to mediate neuronal protection from a wide range of toxic insults and conditions (
      • Brunet A.
      • Datta S.R.
      • Greenberg M.E.
      ). In many cases, specific inhibition of PI3K (e.g. wortmannin and LY294002) has been instrumental in establishing the neuroprotective action of this pathway (
      • Zheng W.H.
      • Kar S.
      • Quirion R.
      ). However, we report here that transient inhibition of the PI3K-Akt pathway can protect primary neurons from oxidative toxicity. Thus, PI3K activity may be required for some initial steps of a cell death program that unfolds when neurons are subjected to oxidative stress. To our knowledge, this is the first demonstrated example of PI3K activity associated with cell death. Importantly, a transient “window” of PI3K inhibition in mixed cortical cultures exposed to LY294002 or wortmannin enabled us to distinguish between the short term and long term requirements for PI3K activity in this neurotoxicity model. Transient PI3K inhibition is apparently insufficient to undermine the long term pro-survival function of this pathway. Because neuron-enriched cultures of the same age treated with LY294002 do not exhibit a transient inhibition of PI3K activity, it seems likely that glial cells in our cultures (∼20–25%) are responsible for the metabolism, inactivation, or sequestration of LY294002. Although we have not detected appreciable inactivation of LY294002 in enriched mature astrocyte cultures (data not shown), there are other glial cell types in our mixed cultures that could exhibit this activity.
      Our results are consistent with the wealth of data supporting a neuroprotective role for the PI3K pathway, including an established cell culture model of oxidative toxicity (i.e. HT22 cells). We were able in our mixed cultures to generate prolonged inhibition of PI3K by simply administering LY294002 in two boluses 6 h apart. In this case where PI3K activity was inhibited for prolonged periods of time (i.e. up to 12 h), the neuroprotective effects of LY294002 were abrogated. Furthermore, in neuron-enriched cultures, a single bolus of LY294002 was toxic because of prolonged inhibition of the PI3K-Akt pathway (Fig. 4C). This finding is consistent with recent data showing that a single 20 μm dose of LY294002 is toxic to neuron-enriched primary hippocampal cultures (
      • Luo H.
      • Hattori H.
      • Hossain M.
      • Hester L.
      • Huang Y.
      • Lee-Kwon W.
      • Donowitz M.
      • Nagata E.
      • Snyder S.
      ).
      Transient inhibition of the PI3K-Akt pathway does not affect neuronal survival in other models where prolonged inhibition would be predicted to lead to cellular demise. For example, infusion of the PI3K inhibitor wortmannin, an unstable compound in aqueous solution, into the amygdala of rats impaired their ability to learn in a fear-conditioning paradigm soon after the administration of the drug but did not affect their ability to be effectively re-trained in the same paradigm several days later (
      • Lin C.H.
      • Yeh S.H.
      • Lu K.T.
      • Leu T.H.
      • Chang W.C.
      • Gean P.W.
      ). This implies that the effects of wortmannin in vivo are reversible and do not result in neuronal cell death, at least in the amygdala. Similarly, LY294002 has been used in vivo in models of transient focal cerebral ischemia- or seizure-induced neuronal toxicity and, in these studies, LY294002 alone did not lead to increased neuronal cell death in the hippocampus or the caudate/putamen (
      • Noshita N.
      • Lewen A.
      • Sugawara T.
      • Chan P.H.
      ,
      • Henshall D.C.
      • Araki T.
      • Schindler C.K.
      • Lan J.Q.
      • Tiekoter K.L.
      • Taki W.
      • Simon R.P.
      ), presumably because of the surrounding glia. Our demonstration of transient PI3K inhibition by LY294002 in mixed neuronal/glial cultures provides strong support for the notion that metabolism, inactivation, or sequestration of LY294002 can have functional consequences for neuronal cell responses to a cell death-inducing stimulus. This would be of great interest in the development of therapeutic uses of these compounds in vivo where surrounding glial elements would probably alter the pharmacodynamics of these drugs in the central nervous system.
      We have previously determined that glutamate-induced oxidative toxicity is associated with the development of a delayed activation of ERK1/2 and that the MEK inhibitor prevents this activation and is significantly neuroprotective (
      • Stanciu M.
      • Wang Y.
      • Kentor R.
      • Burke N.
      • Watkins S.
      • Kress G.
      • Reynolds I.
      • Klann E.
      • Angiolieri M.R.
      • Johnson J.W.
      • DeFranco D.B.
      ,
      • Stanciu M.
      • DeFranco D.B.
      ). Interestingly, the ability of transient PI3K inhibition to protect these cultures is associated with an abrogation of delayed MEK-1 activation (Fig. 6) and ERK1/2 phosphorylation (Fig. 5). This further establishes ERK1/2 activation in this model as a requisite event for eventual cellular demise. In addition, because of the temporal disparity between the period of PI3K inhibition and the regulation of ERK1/2 activity, we can place PI3K activation upstream of the course of events that leads to MEK-1 and ERK1/2 activation. Furthermore, the fact that neither PI3K nor MEK inhibition blocks oxidative stress implies that somehow PI3K inhibition can uncouple ERK activation from oxidative stress and that oxidative stress is not dependent upon ERK activation in this model (Fig. 7).
      Cross-talk between the PI3K-Akt and Raf-MEK-ERK pathways has been demonstrated in an increasingly diverse set of circumstances and in different cell types. Although this link was first reported to be involved in the signaling of G-protein coupled receptors (
      • Hawes B.E.
      • Luttrell L.M.
      • van Biesen T.
      • Lefkowitz R.J.
      ,
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ), PI3K activity can be necessary for the recruitment of the ERK pathway in signaling events associated with other types of receptors as well. For example, PI3K activity is required for in vitro ERK activation in AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) and NMDA (N-methyl d-aspartate) receptor-mediated signaling events in striatal cultures (
      • Perkinton M.S.
      • Sihra T.S.
      • Williams R.J.
      ,
      • Perkinton M.S.
      • Ip J.K.
      • Wood G.L.
      • Crossthwaite A.J.
      • Williams R.J.
      ) and for the nerve growth factor-induced sustained activation of ERK during PC12 differentiation (
      • York R.D.
      • Molliver D.C.
      • Grewal S.S.
      • Stenberg P.E.
      • McCleskey E.W.
      • Stork P.J.
      ). Furthermore, PI3K activation was shown to occur upstream of ERK activation in a rat model of associative learning (
      • Lin C.H.
      • Yeh S.H.
      • Lu K.T.
      • Leu T.H.
      • Chang W.C.
      • Gean P.W.
      ). Of great interest is the recent finding that PI3K positively contributes to the activation of ERK1/2 in cortical cultures exposed to oxidative stress induced by hydrogen peroxide (
      • Crossthwaite A.J.
      • Hasan S.
      • Williams R.J.
      ). Thus, there appears to exist a mechanism for functional coupling of the PI3K and ERK pathways in neurons with the potential for PI3K to act as a required upstream activator.
      Contradictory effects of PI3K on the ERK pathway have been noted and were initially attributed to cell type-specific signaling events that reflected a wide variety of direct and indirect interactions between the two pathways (
      • Hawes B.E.
      • Luttrell L.M.
      • van Biesen T.
      • Lefkowitz R.J.
      ,
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ,
      • Zimmermann S.
      • Moelling K.
      ). However, recent reports showing opposing effects of PI3K on ERK activation within a single cell type in response to one signal reveal the importance of signal intensity in dictating the eventual outcome of PI3K/ERK cross-talk (
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Wennstrom S.
      • Downward J.
      ,
      • Moelling K.
      • Schad K.
      • Bosse M.
      • Zimmermann S.
      • Schweneker M.
      ). Furthermore, EGF-induced ERK activation was found to be PI3K-dependent under conditions where PI3K activity remained near basal levels (
      • Wennstrom S.
      • Downward J.
      ). This previously described “permissive” effect of the PI3K pathway on ERK activation (
      • Crossthwaite A.J.
      • Hasan S.
      • Williams R.J.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Wennstrom S.
      • Downward J.
      ,
      • Sutor S.L.
      • Vroman B.T.
      • Armstrong E.A.
      • Abraham R.T.
      • Karnitz L.M.
      ) appears to operate in our neuronal cultures because increased stimulation of PI3K activity in response to glutamate treatment is minimal (data not shown). However, our neuronal cultures do exhibit a substantial base-line level of PI3K activity (Figs. 3, A and B, and 4A). Interestingly, our results are unique in that the coupling between PI3K and ERK is restricted to a distinct kinetic phase during the progression of an oxidative stress-induced cell death pathway. Thus, the inhibition of basal PI3K activity (as assessed by levels of phosphorylated Akt) at early times following the initiation of glutamate treatment (i.e. within 4–6 h) impacts ERK activation that is only apparent following an additional 4–6 h.
      We consider the observed coupling in our primary neuron cultures to reflect a permissive effect of PI3K on ERK activation. Glutamate-induced oxidative toxicity in immature neurons and in HT22 cells is driven by a delayed rise in intracellular ROS and Ca2+ (
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ). The rapid burst and accumulation of ROS and Ca2+ that ultimately results from glutamate treatment are likely to be proximal to the terminal execution phases of the cell death program that operates in these cells (
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ). PI3K has been found to influence plasma membrane Ca2+ channel activity through activated Akt and thereby contribute to neuronal cell survival in response to neuroprotective factors such as insulin-like growth factor-1 (
      • Blair L.A.
      • Bence-Hanulec K.K.
      • Mehta S.
      • Franke T.
      • Kaplan D.
      • Marshall J.
      ). Furthermore, a voltage-dependent calcium channel-dependent form of long term potentiation at hippocampal CA1 region synapses was found to require PI3K activity, implying that PI3K activity can directly regulate calcium entry into neurons (
      • Sanna P.P.
      • Cammalleri M.
      • Berton F.
      • Simpson C.
      • Lutjens R.
      • Bloom F.E.
      • Francesconi W.
      ). In addition, PI3K has been demonstrated to be required for the activation of voltage-independent Ca2+ channels in a Chinese hamster ovary cell system (
      • Kawanabe Y.
      • Hashimoto N.
      • Masaki T.
      ) and voltage-dependent calcium channels in vascular smooth muscle cells (
      • Seki T.
      • Yokoshiki H.
      • Sunagawa M.
      • Nakamura M.
      • Sperelakis N.
      ), non-selective cation channels (
      • Kawanabe Y.
      • Hashimoto N.
      • Masaki T.
      ), and for Ca2+ release from intracellular store-operated calcium channels (
      • Kawanabe Y.
      • Hashimoto N.
      • Masaki T.
      ). Furthermore, there is increasing evidence showing that PI3K activity is not only necessary for increases in intracellular Ca2+ but that this increase in Ca2+ is necessary for ERK activation (
      • Kansra V.
      • Groves C.
      • Gutierrez-Ramos J.
      • Polakiewicz R.
      ). Interestingly, PI3K-dependent increases in intracellular Ca2+ have been shown to be dissociated from increases in phosphorylated Akt and may represent a point of divergence between action of PI3K on the Akt versus the ERK pathway (
      • Kawanabe Y.
      • Hashimoto N.
      • Masaki T.
      ,
      • Kansra V.
      • Groves C.
      • Gutierrez-Ramos J.
      • Polakiewicz R.
      ). This is consistent with our findings that Akt activation (above base line) is minimal during the development of glutamate-induced oxidative toxicity (data not shown). Thus, it is tempting to speculate that the permissive effects of PI3K on ERK-dependent cell death in oxidatively stressed primary neurons might be attributed to the effects of basal PI3K activity on either plasma membrane Ca2+ channels or intracellular store-operated channels. ROS generated by the mitochondria contribute to Ca2+ influx and accumulation in glutamate-treated HT22 cells and primary cortical neurons (
      • Li Y.
      • Maher P.
      • Schubert D.
      ,
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ), and a Ca2+ channel response to ROS that requires some direct or indirect priming action of the PI3K pathway may very well be the link between the PI3K and ERK pathways in our system.
      The identification of multiple intracellular signaling pathways that operate to promote or limit neuronal cell death and the delineation of various levels of cross-talk between these pathways both present unique challenges to the development of pharmacological neuroprotective therapies. Our results suggest that depending upon the nature and identity of the coupled signal transduction pathways, a temporal window may exist that allows for the transient inhibition of a specific signal transduction pathway. Thus, rapid metabolism or sequestration of drugs targeted to specific signaling pathways may prove beneficial particularly in cases where unique coupling between signal transduction pathways occurs at distinct kinetic phases of a progressing cell death program.

      Acknowledgments

      We thank Dr. Julie Pongrac for excellent training and advice with the primary neuronal cultures. Dr. Sophie Lee is also thanked for preliminary data demonstrating the toxicity of LY294002 in HT22 cells.

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