Glutamate excitotoxicity attenuates insulin-like growth factor-i prosurvival signaling

Molecular and Cellular Neuroscience 24 (2003) 1027–1037

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Glutamate excitotoxicity attenuates insulin-like growth factor-I prosurvival signaling E. Garcia-Galloway, C. Arango,1 S. Pons,2 and I. Torres-Aleman* Laboratory of Neuroendocrinology, Cajal Institute, CSIC, Avda. Dr. Arce 37. 28002 Madrid, Spain Received 9 April 2003; revised 18 July 2003; accepted 7 August 2003

Abstract Recent evidence suggests that impaired insulin/insulin-like growth factor I (IGF-I) input may be associated to neurodegeneration. Several major neurodegenerative diseases involve excitotoxic cell injury whereby excess glutamate signaling leads to neuronal death. Recently it was shown that glutamate inactivates Akt, a serine-kinase crucially involved in the prosurvival actions of IGF-I. We now report that excitotoxic doses of glutamate antagonize Akt activation by IGF-I and inhibit the neuroprotective effects of this growth factor on cultured neurons. Glutamate induces loss of sensitivity to IGF-I by phosphorylating the IGF-I receptor docking protein insulin-receptor-substrate (IRS)-1 in Ser307 through a pathway involving activation of PKA and PKC in a hierarchical fashion. Administration of Ro320432, a selective PKC inhibitor, abrogates the inhibitory effects of glutamate on IGF-I-induced Akt activation in vitro and in vivo and is sufficient to block the neurotoxic action of glutamate on cultured neurons. Notably, administration of Ro320432 after ischemic insult, a major form of excitotoxic injury in vivo, results in a marked decrease (⬃50%) in infarct size. Therefore, uncoupling of IGF-I signaling by glutamate may constitute an additional route contributing to excitotoxic neuronal injury. Further work should determine the potential use of PKC inhibitors as a novel therapeutic strategy in ischemia and other excitotoxic insults. © 2003 Elsevier Inc. All rights reserved.

Introduction Insulin-like growth factor I (IGF-I) blocks neuronal death after a wide variety of insults, including those of ample impact, such as hypoxia or oxidative stress, and others more specific such as amyloid-␤ toxicity or neurotoxic injury (Torres-Aleman, 2000). Levels of IGF-I are upregulated in brain lesions, regardless of their type (Dore et al., 1997), further suggesting that this neurotrophic factor exerts a wide-spectrum role in neuronal protection. IGF-I may counteract such an ample array of neurodegenerative processes through modulation of common death pathways, including programmed cell death (Singleton et al., 1996) or energy imbalances (Fernandez et al., 1998). Alternatively, IGF-I may exert a tonic protection on neurons and its loss may lead to cell damage and eventual neuronal demise. In * Corresponding author. Fax: ⫹3491-585-4754. E-mail address: [email protected] (I. Torres-Aleman). 1 Present address: Universidad del Valle, Cali, Colombia. 2 Present address: Institute of Biomedicine, CSIC, Barcelona, Spain. 1044-7431/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.08.005

this view, deleterious signals will diminish the ability of a neuron to respond to IGF-I; i.e., neurons will become resistant to the actions of IGF-I as is the case for insulinsensitive cells in type 2 diabetes. This possibility is particularly plausible for several reasons. If damaged neurons loss sensitivity to IGF-I, a normal compensatory homeostatic mechanism would be to increase the levels of IGF-I, as seen in brain lesions (Dore et al., 1997), in an attempt to regain normal trophic input (Jain et al., 1998). Furthermore, at least one cytotoxic signal, tumor necrosis factor-␣ (TNF-␣), which induces insulin/IGF-I resistance in several tissues (Hotamisligil et al., 1996), has been found to counteract IGF-I signaling also in the brain (Venters et al., 1999; Carro et al., 2002). Other neurotoxic signals related to loss of neuronal sensitivity to IGF-I include such diverse agents as prion proteins, ethanol, and the toxin methylmercury (Bulleit and Cui, 1998; Zhang et al., 1998; Ostlund et al., 2001). Resistance to IGF-I may not be circumscribed to sporadic degenerative processes, but may also be involved in inherited diseases such as ataxia-telangiectasia or dentatorubral-pallidoluysian atrophy (Busiguina

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et al., 2000). Both diseases are characterized by reduced expression of proteins involved in insulin/IGF-I signaling (Peretz et al., 2001). Therefore, neuronal death in these diseases may be related to inappropriate insulin/IGF-I trophic signaling. We now have explored whether loss of sensitivity to the prosurvival actions of IGF-I is a common feature of damaged neurons. For this purpose we have analyzed excitotoxic neuronal damage due to excess glutamate signaling because excess glutamatergic input is thought to be involved in pathological conditions of great impact such as stroke, amyotrophic lateral sclerosis, epilepsia, or brain and spinal cord trauma (Beal, 1992). Glutamate has recently been shown to inhibit the kinase Akt (Chalecka-Franaszek and Chuang, 1999), which is essential for the prosurvival actions of IGF-I (Dudek et al., 1997). We describe a novel pathogenic pathway linking excess glutamate input to down-regulation of IGF-I prosurvival signaling through Akt. This pathway appears to be active in ischemic damage because its inhibition results in diminished lesion size.

Results Glutamate blocks phosphorylation of Akt by IGF-I

Fig. 1. Glutamate antagonizes IGF-I. (A) Excitototoxic levels of glutamate disrupt IGF-I-induced neuronal survival. (Upper photographs) Phase-contrast micrograph of cerebellar cultures used to analyze interactions between glutamate and IGF-I on cell survival (left panel); fluorescence photomicrograph of propidium iodide-stained neurons in control (center) and glutamate-treated cultures (right). Note the marked increase in dead cells after excitotoxic insult. (Histograms) Quantification of living cells after 500 ␮M glutamate and/or 100 nM IGF-I. *P ⬍ 0.005 vs IGF-I alone (n ⫽ 6). (B, upper panel) Thirty minutes after adding IGF-I (100 nM) phosphorylation of Akt (pAKt, upper gel) is stimulated in cerebellar cultures, an effect antagonized by glutamate (500 ␮M). Total Akt levels (lower gel) are not modified. (B, lower panel) Quantitative densitometric analysis of Western

IGF-I exerts prosurvival effects on neurons (D’Ercole et al., 1996), whereas excess glutamate is neurotoxic (Beal, 1992). This opposing action of IGF-I and glutamate on cell survival is evidenced when neurons grown in the presence of IGF-I are challenged with a cytotoxic dose of glutamate (500 ␮M): enhanced cell survival after IGF-I (100 nM) is blunted by glutamate (Fig. 1A). Antagonistic effects of glutamate on IGF-I actions are found for a broad range of doses, i.e., the lowest dose at which glutamate is neurotoxic: 100 ␮M is able to block the trophic effects of IGF-I at all doses used (1–100 nM; not shown). We then explored pathways involved in the antagonism between IGF-I and glutamate and focused on the serine-kinase Akt because glutamate inactivates this kinase in neurons (Chalecka-Franaszek and Chuang, 1999) and, at the same time, activation of Akt by IGF-I is critical for its prosurvival effects (Dudek et al., 1997). Hence, Akt appears as a likely target for possible glutamate-IGF-I interactions. IGF-I stimulates phosphorylation of Akt in cerebellar neurons, an effect antagonized by glutamate (Fig. 1B, P ⬍ 0.05). Although it is well documented that IGF-I stimulates Akt phosphorylation through the IRS/PI3K pathway (Dudek

blots. Results shown are levels of pAkt after normalizing for levels of Akt. *P ⬍ 0.05 vs IGF-I alone (n ⫽ 6). (C) Inhibition of phosphatases does not affect glutamate-induced inhibition of IGF-I-stimulated pAkt. Coaddition of okadaic acid (Okad, 50 nM), cyclosporin (Cyclos, 200 ␮M), or BAPTA-AM (200 ␮M) did not restore low pAkt levels after glutamate. *P ⬍ 0.05 vs control (n ⫽ 4). In this and following figures, C, controls; I, IGF-I; G, glutamate.

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et al., 1997), the route involved in dephosphorylation of Akt by glutamate is not known, although an as-yet-undetermined phosphatase has been proposed (Chalecka-Franaszek and Chuang, 1999). Therefore, modulation of Akt activity by IGF-I and glutamate may involve either independent or common signaling pathways (Chalecka-Franaszek and Chuang, 1999). We determined which of these two possibilities underlies opposing regulation of Akt activity by IGF-I and glutamate in neurons. We tested phosphatase inhibitors such as okadaic acid (0.5 nM and 50 nM to inhibit protein phosphatase-1 and -2, respectively), tautomycin (10 nM to inhibit protein phosphatase-2), cyclosporin (200 ␮M to inhibit calcineurin), or the calcium chelator BAPTA-AM (200 ␮M) to determine if the inhibotory effects of glutamate on Akt phosphorylation induced by IGF-I could be blocked. As shown in Fig. 1C, okadaic acid at 0.5 nM, cyclosporin, or BAPTA-AM do not interfere with glutamate. Similar lack of effect was found with the other drugs and doses (not shown). Thus, we considered the possibility that glutamate deactivates IGF-I-induced Akt phosphorylation by directly interfering with IGF-I signaling. We first analyzed whether glutamate modulates phosphorylation of the IGF-I receptor by IGF-I and found that IGF-I phosphorylates it regardless of the presence of glutamate (Fig. 2A). A possible way to disrupt insulin/IGF-I signaling is by interfering with IRS activation by the IGF-I receptor (Kanety et al., 1995). As shown in Fig. 2B, glutamate counteracts IGF-I-induced Tyr phosphorylation of IRS-1 (P ⬍ 0.05 vs IGF-I alone). Downstream signaling through IRS-1 includes association of PI3Kp85 to activated IRS (Pons et al., 1995). We measured association of PI3Kp85 to Tyr-phosphorylated IRSs by immunoprecipitating cell lysates with anti-PI3Kp85 and immunoblotting with anti-pTyr or anti-IRS-1 antibodies. In this way, levels of pTyrIRSs associated to PI3Kp85 can be monitored. Figure 2C (upper blot) shows that glutamate markedly reduces association of IRS-1 to PI3Kp85 induced by IGF-I as well as the amount of pTyr (band at 185 kDa corresponding to the size of IRS-1) associated to PI3Kp85 (lower blots). Because in our cultures IRS-2 levels are barely detectable (not shown), it is very likely that IGF-I signaling involves mostly IRS-1 activation. Fig. 2. Glutamate interferes with IGF-I signaling through inhibition of the IRS-1/PI3K pathway. (A) Phosphorylation of the IGF-I receptor by 100 nM IGF-I (3-min stimulation) is not affected by 500 ␮M glutamate. (A, upper panel) Representative anti-pTyr blot of IGF-I receptor immunoprecipitates. Total amount of IGF-I receptor was similar in all treatments (lower blot). (A, lower panel) Densitometric analysis (n ⫽ 5). (B) After 3 min of stimulation with IGF-I Tyr-phosphorylated IRS-1 is increased, an effect antagonized by glutamate. (B, upper panel) Blot with anti-pTyr of IRS-1 immunoprecipitates. Amount of immunoprecipitated IRS-1 is not affected by treatments (lower blot). (B, lower panel) Densitometry after normalization of pTyr-IRS-1 for total IRS-1 levels. *P ⬍ 0.05 vs IGF-I alone (n ⫽ 5). (C) Glutamate inhibits stimulation by IGF-I of the association of pTyr-IRS with PI3K regulatory subunit p85 (PI3K). Cultures were stimulated for 3 min. (C, upper blots) IRS-1 blot with PI3K immunoprecipitates show that treatment with glutamate diminishes IGF-I-induced association of IRS-1 to PI3K. (C, lower blots) Immunoblot of pTyr in PI3K immunoprecipitates confirms that association of activated IRS (pTyr-IRS)

Glutamate inhibits IGF-I signaling through PKA and PKC Phosphorylation of IRS in serine residues uncouples its association to the activated insulin/IGF-I receptor leading to cellular resistance to these hormones (Rui et al., 2001). Glutamate signaling through both ionotropic and metabo-

to PI3K is decreased by glutamate. Total amount of immunoprecipitated PI3K is similar after all treatments (lower blots). (C, lower panel) Densitometry shows a significant reduction by glutamate of IGF-I-induced association of pTyr-IRSs to PI3Kp85. *P ⬍ 0.01 vs IGF-I alone (n ⫽ 5).

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tropic receptors potentially recruits different Ser-kinases (Skaper et al., 2001). To determine whether activation of a Ser-kinase underlies dissociation of IRS-1 to the IGF-I receptor after glutamate we used a wide variety of inhibitors, including U73122 (10 ␮M) to inhibit PLC␥; H89 (1 ␮M) to inhibit PKA; bisindolylmaleimide I (100 nM, a wide-spectrum PKC inhibitor); PP2 (50 nM, an inhibitor of src-kinases); SB203580 (10 ␮M, an inhibitor of p38MAPK); KN-93 (15 ␮M) and K-252a (40 nM), both inhibitors of CaM kinases; the PI3K inhibitors LY94002 (25 ␮M) and wortmannin (100 nM); and the MEK1 inhibitor PD98059 (25 ␮M). We found that only H89 and bisindolylmaleimide block glutamate inhibition of IRS/PI3K association (Fig. 3A). Although glutamate induces a significant decrease in the IGF-I-induced association of PI3K to pTyrIRS-1 (from a 2-fold increase after IGF-I alone to a 1.3-fold increase after glutamate ⫹ IGF-I, P ⬍ 0.05), coaddition of either H89 or bisindolylmaleimide abolishes glutamate inhibition (Fig. 3A, n ⫽ 5). Similarly, conjoint exposure to either H89 or bisindolylmaleimide abrogates glutamate-induced inhibition of Akt phosphorylation after IGF-I (representative blot in Fig. 3B; 207% increase in pAkt levels after IGF-I down to 137% stimulation after IGF-I ⫹ glutamate, compared to 198 and 196% increase after IGF-I ⫹ glutamate ⫹ H89 or IGF-I ⫹ glutamate ⫹ bisindolylmaleimide, respectively; P ⬍ 0.05; n ⫽ 5). Drugs targeting the p42– 44MAPK-, p38MAPK-, src-kinase-, PLC-␥-, or CamKs-dependent pathways were ineffective (not shown). Glutamate excitotoxity has been reported to involve activation of Jun-kinase (JNK, Schwarzschild et al., 1997) and in turn JNK can phosphorylate IRS-1 in serine residues (Rui et al., 2001). However, as seen with other types of excitotoxic insults in cerebellar cells (Gunn-Moore and Tavare, 1998), no changes in phospho-JNK levels are found after glutamate treatment of our cultures (Fig. 3C). To confirm that stimulation of PKC and/or PKA is involved in the effects of glutamate on IGF-I signaling we added together with IGF-I either TPA (100 nM), a widespectrum stimulator of PKCs or forskolin (10 ␮M), a potent stimulator of PKA. Both drugs are able to mimic the action of glutamate: IGF-I-induced association of PI3K to pTyrIRS is inhibited by them (Fig. 3D and E; P ⬍ 0.05 vs IGF-I alone for both TPA-IGF-I and forskolin ⫹ IGF-I, n ⫽ 4). Furthermore, inhibition of TPA-stimulated PKC by bisindolylmaleimide disrupts the action of TPA (Fig. 3D, n ⫽ 4). Similarly, inhibition of forskolin-stimulated PKA by conjoint addition of H89 blocks its ability to mimic the effect of glutamate (Fig. 3E, n ⫽ 4). Because coaddition of H89 and bisindolylmaleimide did not further inhibit the action of glutamate (not shown), we consider likely that both kinases converge within a same signaling pathway; i.e., a hierarchical rather than a simultaneous activation of PKA and PKC by glutamate would be involved. To test this, we combined inhibitors and stimulators of PKA and PKC and examined how they affect IGF-I activation of the IRS/ PI3K association. When PKC is inhibited with bisindolyl-

maleimide and PKA is stimulated with forskolin, enhanced association of pTyrIRS to PI3K by IGF-I is not affected (Fig. 3F). However, when PKA is inhibited with H89, activation of PKC with TPA still disrupts increased pTyrIRS/PI3K association after IGF-I (Fig. 3F; P ⬍ 0.05, n ⫽ 5). Therefore, PKA needs an intact PKC activity to block IGF-I signaling, whereas PKC activation mimics the effect of glutamate even in the absence of PKA input. Hence, stimulation of PKA precedes activation of PKC. To start defining specific isoforms of PKC involved in the inhibitory action of glutamate on IRS activation we used isoform-specific inhibitory compounds. Because the actions of glutamate on IGF-I signaling are Ca2⫹-independent (the Ca2⫹ chelator BAPTA-AM does not interfere with them), we used Ro320432, a dose-dependent selective inhibitor of the Ca2⫹-dependent PKC isoforms ␣ and ␤II that also inhibits the novel, Ca2⫹-independent ␧ isoform. As shown in Fig. 3G, uncoupling of IRS-1 to PI3K by glutamate was fully abrogated when 100 nM Ro320432 was added together with glutamate and IGF-I (P ⬍ 0.05 vs IGF-I ⫹ glutamate; n ⫽ 4). A dose of 50 nM Ro320432, which inhibits both PKC␣ and PKC␤II, was ineffective (Fig. 3G). A similar effect was observed on IGF-I-induced Akt phosphorylation; although 10 and 50 nM doses of Ro320432 tend to inhibit the effects of glutamate, only the 100 nM dose was significantly effective (Fig. 3H, P ⬍ 0.05 vs glutamate ⫹ IGF-I, n ⫽ 4). To determine whether PKC␧ is involved in the effects of glutamate we assessed PKC␧ phosphorylation in Ser729 after glutamate challenge. Only excitotoxic doses of 500 ␮M glutamate, but not the nontoxic dose of 5 ␮M, increased pSer729-PKC␧ (Fig. 4A; P ⬍ 0.05 vs control; n ⫽ 4). Phosphorylation of this serine residue is necessary to activate this kinase (Cenni et al., 2002). Phosphorylation of PKC␧ by 500 ␮M glutamate is abrogated by Ro320432 at 100 nM (P ⬍ 0.05; n ⫽ 4). Furthermore, activation of PKC␧ by 500 ␮M glutamate requires PKA activity because H89 inhibits it (Fig. 4A). The next step was to determine whether glutamate promotes phosphorylation of IRS-1 in a PKA- and PKC-dependent fashion in residues such as Ser307, which interfere with the coupling of IRS-1 to the IGF-I receptor (Aguirre et al., 2002). We found that glutamate induces phosphorylation of IRS-1 in Ser307 in a dose-dependent manner (Fig. 4B; left blot). The effect of glutamate on Ser307 phosphorylation depends on PKC␧, because addition of 100 nM Ro320432 abrogates it (Fig. 4B, right gel), and on PKA, because H89 also impedes glutamate-induced phosphorylation of IRS-1 (Fig. 4B). In vivo interference of IGF-I signaling by glutamate We first analyzed whether stimulation of PKC is involved in the neurotoxic effects of glutamate in cultured cells. Indeed, Ro320432 (100 nM) abrogates glutamate toxicity on neuronal cultures (Fig. 5A, P ⬍ 0.05 vs glutamate, n ⫽ 4). To see if this pathway is also involved in the toxicity

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Fig. 3. Glutamate disrupts IGF-I signaling through PKA and PKC. (A) Inhibition of either PKA or PKC abrogates effects of glutamate on IGF-I-induced association of PI3K to pTyr-IRS. (A, upper panel) Representative pTyr blot of PI3K immunoprecipitates showing that H89 or bisindolylmaleimide (Bis) disrupt the inhibitory effects of glutamate on increased PI3K associated to pTyr-IRS after 3 min of IGF-I (P ⬍ 0.05 vs glutamate ⫹ IGF-I for both drugs, n ⫽ 5). Total PI3K levels are not modified (lower blot). Neither H89 nor Bis alone altered levels of pTyrIRS associated to PI3K as compared to control levels (center and rightmost blots). (B) H89 and Bis abrogate the effects of glutamate on activation of Akt by IGF-I. Cultures were stimulated for 30 min. (B, upper panel) pAkt blot showing that H89 or Bis disrupt the inhibitory effects of glutamate on levels of pAkt after IGF-I (P ⬍ 0.05; n ⫽ 5 independent experiments). Total Akt is unchanged (lower blot). (C) Glutamate does not modify phosphorylation of Jun kinase (JNK) in cerebellar cultures. No changes in baseline pJNK were seen after any of the treatments. (C, upper blot) Representative blot of pJNK. (C, lower blot) Total levels of JNK. (Histograms) Quantification showed no differences in pJNK relative to total JNK in any group (n ⫽ 3). (D) Activation of PKC mimics the effects of glutamate. (D, upper panel) Representative pTyr blot of PI3K immunoprecipitates shows that TPA inhibits the stimulatory action of IGF-I on PI3K-associated pTyr-IRS (P ⬍ 0.05 vs IGF-I alone; 3 min stimulation), whereas Bis abrogates TPA effects (P ⬍ 0.05 vs IGF-I ⫹ TPA; n ⫽ 4). Total PI3K is not affected (lower blot). (Right blot) TPA alone does not alter control levels. (E) Representative pTyr blot of PI3K immunoprecipitates illustrating that forskolin (Forsk) inhibits IGF-I-induced increase in pTyr-IRS associated to PI3K (P ⬍ 0.05 vs IGF-I alone). H89 abrogates forskolin effects (P ⬍ 0.05 vs IGF-I ⫹ Forsk; n ⫽ 4). Forskolin alone does not alter levels of pTyr-IRS associated to PI3K (right blot). (F) Activation of PKC while PKA is inhibited mimics the inhibitory effects of glutamate. Activation of PKA in the presence of inhibited PKC does not mimic glutamate. (F, upper panel) Representative pTyr blot of PI3K immunoprecipitates show that the combined action of TPA ⫹ H89 reduces IGF-I-induced association of pTyr-IRS to PI3K, whereas Bis ⫹ Forsk does not affect the response to IGF-I. (Histograms) A significant reduction in pTyr-IRS associated to PI3K is seen in TPA ⫹ H89 ⫹ IGF-I-treated cultures. *P ⬍ 0.05 vs all other groups (n ⫽ 5). (G) Coaddition of 100 nM, but not 50 nM, Ro320432 (Ro) blocks glutamate inhibition of the association of IRS-1 to PI3K after IGF-I (P ⬍ 0.05 vs glutamate ⫹ IGF-I, n ⫽ 4). (F, upper panel) IRS-1 immunoblot of PI3K immunoprecipitates. Levels of PI3K are unaffected (lower panel). (Histograms) Quantitation of immunoblots. (H) Ro320432 inhibits in a dose-dependent manner dephosphorylation of Akt by glutamate. At doses of 10 and 50 nM, Ro320432 fully inhibits PKC␣ and ␤II, respectively, whereas at 100 nM inhibits also PKC␧ (Birchall et al., 1994). (H, upper panel) Representative pAkt blot. Total Akt is unchanged (lower blot). (H, lower panel) Quantitative assessment of the inhibitory action of Ro320432. *P ⬍ 0.05 vs glutamate ⫹ IGF-I; n ⫽ 4.

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brain pAkt levels (Fig. 5B), coadministration of glutamate ⫹ PCDA (a glutamate reuptake inhibitor) abrogates peak levels of pAkt after 1 h of IGF-I (Fig. 5C). As in cell cultures, Ro320432 ameliorates the inhibitory effects of glutamate on brain pAkt levels 1 h after IGF-I (Fig. 5D) and blocks the glutamate-induced Ser phosphorylation of IRS-1 seen 10 min after administration of glutamate (Fig. 5E). To start delineating a possible biological significance of these observations we examined whether excitotoxic injury in ischemic insults (Beal, 1992), a major form of acute neurodegenerative disease, is modulated by Ro320432. We administered a chronic icv infusion of Ro320432 (100 mM) immediately after rats were subjected to transient (90 min) medial cerebral artery occlusion. A week later, infarct size was determined and found to be drastically reduced (50%) in Ro320432-infused animals as compared to saline-treated animals (Fig. 5F, P ⬍ 0.05, n ⫽ 5).

Discussion

Fig. 4. Glutamate activates PKC␧ and phosphorylates IRS-1 in Ser307. (A) A 3-min stimulation with high (500 ␮M), but not low (5 ␮M), glutamate stimulates phosphorylation of Ser709 PKC␧. The effect of 500 ␮M glutamate is abrogated by Ro (100 nM) and depends on PKA activity because H89 counteracts it. Levels of total PKC (pan-pecific PKC antibody) are not altered. (Histograms) Quantitation of pSer-PKC␧ after correcting for total PKC. *P ⬍ 0.05 vs control, n ⫽ 4. (B) A 3-min exposure to glutamate stimulates phosphorylation of Ser307 IRS-1 through PKC activation. (Upper panels) Glutamate phosphorylates IRS-1 in a dose-dependent manner. (Lower panels) Phosphorylation of IRS-1 in Ser307 by high doses of glutamate is inhibited by 100 nM Ro. Similarly, inhibition of PKA inhibits the effects of 500 ␮M glutamate. Total IRS-1 levels are not altered. (Histograms) Densitometric quantitation of pSerIRS-1. *P ⬍ 0.05 vs control, n ⫽ 3.

of glutamate in brain we examined whether glutamate interferes with IGF-I signaling in vivo and whether Ro320432 modulates the actions of glutamate. We assessed brain levels of pAkt as a determinant of the actions of IGF-I because activation of this kinase has been shown to mediate neuroprotection by IGF-I (Dudek et al., 1997). Although icv administration of IGF-I elicits a time-dependent increase in

It has previously been shown that the prosurvival actions of IGF-I involve activation of the PI3K/Akt pathway (Dudek et al., 1997). The present results indicate that excitotoxic levels of glutamate interfere with this neuroprotective pathway. Together with recent observations of loss of sensitivity to insulin/IGF-I in certain types of neurodegenerative conditions (Venters et al., 1999; Ostlund et al., 2001; Peretz et al., 2001) our findings support a new notion whereby cytotoxic signals may operate by interfering with neurotrophic pathways. In this interpretation, excess glutamate is neurotoxic also because it attenuates trophic IGF-I signaling. Although this may appear to contradict previous extensive research indicating a role for intracellular Ca2⫹ in glutamate excitotoxicity (Sattler and Tymianski, 2000), our proposal can be easily reconciled with these findings by considering loss of sensitivity to IGF-I as an additional route in glutamate-induced neuronal death (see Fig. 6). Our results suggest that glutamate blocks IGF-I prosurvival through hierarchical activation of PKA and then of PKC, probably the PKC␧ isoform. In turn, PKC phosphorylates IRS in Ser307 to uncouple it from the activated IGF-I receptor since Ser phosphorylation of IRS impedes its activation by tyrosin-kinases such as the IGF-I or insulin receptors (Aguirre et al., 2002). Indeed, glutamate requires PKA and PKC in a sequential fashion. Further, glutamate phosphorylates IRS-1 in Ser307 through PKA and PKC. Finally, activation of PKC by glutamate requires PKA. Previous work supports this proposed pathway. For example, PKA has been shown to activate PKC (RobinsonWhite and Stratakis, 2002). Other findings show that PKC inhibits Akt phosphorylation through Ser phosphorylation of IRS-1 (Zheng et al., 2000). Further evidence indicate that the multifunctional docking proteins IRSs are targets of insulin/IGF-I resistance-inducing pathways (Paz et al., 1997) through phosphorylation of Ser residues that uncou-

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Fig. 5. Activation of PKC by glutamate in vivo. (A) Inhibition of PKC with 100 nM Ro320432 fully abrogates glutamate (500 ␮M) excitotoxicity on neuronal cultures. Number of living cells after glutamate were reduced ⬃40%, but the presence of Ro inhibited glutamate-induced cell death. *P ⬍ 0.05 vs control, n ⫽ 4. (B) Time course of IGF-I induced increases in hippocampal pAkt. Animals received an icv injection of IGF-I (100 ␮M) and pAkt levels at the injection site (ipsilateral, I) were compared to those in the contralateral (C) uninjected hippocampus at various times thereafter. Peak expression was seen at 1 h; this time was chosen for subsequent experiments. Total levels of Akt are unaffected (lower blot). (C) Coadministration of glutamate (10 mM) ⫹ PDCA (15 mM, an inhibitor of glutamate reuptake) abolishes the increase in brain pAkt measured 1 h after intraventricular injection of IGF-I (100 ␮M). (Histograms) Quantitation of pAkt levels after normalizing for total Akt (lower gel) content. P ⬍ 0.05 vs control (saline) animals, n ⫽ 5. (D) Glutamate-induced decrease in brain pAkt levels after IGF-I is inhibited by administration of Ro (100 ␮M). (Histograms) Quantitation of pAkt normalizing with levels of calbindin (a neuronal marker) indicates abrogation of glutamate effects. P ⬍ 0.05 vs control, n ⫽ 5 animals per group. (E) Increased phosphorylation in Ser307 IRS-1 is seen 10 min after icv administration of glutamate ⫹ PDCA. This increase is inhibited by prior administration of Ro (100 ␮M, 45 min before excitotoxic challenge). (Histograms) Quantitation of pSer307 IRS-1 levels after normalizing for total IRS-1. (E, lower blot) Total IRS-1 levels were not changed by treatments. *P ⬍ 0.05 vs all other groups, n ⫽ 3. (F) Chronic icv administration of Ro (100 mM) immediately after arterial occlusion results in a drastic reduction in infarct volume (⬃50%) determined 7 days after ischemic injury. (Upper photographs) Representative brain sections of saline and Ro-treated animals are shown to illustrate infarct size. P ⬍ 0.05 vs saline treated; n ⫽ 5 rats per group.

ple IRS from the IGF-I receptor (Rui et al., 2001). Although IRSs contain PKC consensus phosphorylation sites, activation of other kinases may also lead to Ser phosphorylation of

IRSs. For example, not only PKCs-␣ or -␧ (Kellerer et al., 1997; Rosenzweig et al., 2002) but also MAPK (Rui et al., 2001) or PI3K (Ozes et al., 2001) are reportedly needed to

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Fig. 6. Excitotoxic pathways to cell death: interference with IGF-I signaling. Excitotoxic doses of glutamate will recruit both ionotropic and metabotropic glutamate receptors leading to activation of PKA and then of PKC (probably PKC␧). The latter may signal through two nonexclusive routes: (1) to increase intracellular Ca2⫹ fluxes resulting in activation of well-described cell-death pathways and (2) To phosphorylate Ser307 in IRS-1. Phosphorylation of this serine residue of IRS-1 impedes its tyr phosphorylation by the activated IGF-I receptor. In this way IRS-1 is not activated and the regulatory subunit of PI3K (p85) does not associate to it. As a result, the catalytic subunit of PI3K (p110) does not activate Akt and prosurvival pathways are interrupted. This route may coexist with other death pathways involving activation of other types of PKC isoforms. In addition, the inhibitory route through PKC␧ can also be recruited by TNF-␣ to phosphorylate IRS-1. However, other kinases have also been implicated in the inhibitory actions of TNF-␣ on IRS-1 (see text).

mediate TNF-␣-induced insulin/IGF-I receptor desensitization. Similarly, not only PKC␨ (Liu et al., 2001) but also PI3K (Rui et al., 2001) have been implicated in insulin/ IGF-I autoregulatory feedback loops. All these agree with our data suggesting that glutamate recruits PKC to phosphorylate IRS-1. Reinforcing this notion is our finding that inhibition of PKC with Ro320432 is sufficient to abolish glutamate excitotoxicity. Broad-spectrum inhibitors of PKC have already been shown to inhibit the excitotoxic actions of glutamate (Favaron et al., 1990). The difference is that in previous studies PKC activation was hypothesized to modulate Ca2⫹ or the NMDA receptor, whereas we provide direct evidence that PKC inhibits IGF-I signaling. Based on the fact that Ro320432 inhibits the action of glutamate only at doses that inhibit PKC␧, the ␣ and ␤II PKC isoforms also inhibited by Ro320432 are Ca2⫹-dependent and the effects of glutamate on IGF-I signaling are not, and glutamate phosphorylates PKC␧ only at high doses, we consider likely that this PKC isoform is mediating the inhibitory actions of excess glutamate on IGF-I signaling. However, detailed molecular studies are required to ascertain this point. Both stimulation of PKA (Beaver et al., 2001) and PKC (Calabresi et al., 2001) have been reported to be downstream of glutamate receptor activation. Simultaneous activation of PKA and PKC has also recently been shown to occur in response to glutamate (Huang et al., 1999; Bandrowski et al., 2001). However, the specific pathways linking

glutamate receptors to activation of PKA/PKC remain to be established, although considerable evidence points to a role for the metabotropic subtype of glutamate receptors (Calabresi et al., 2001), but see (Beaver et al., 2001; Sattler and Tymianski, 2001) and PKC␧ is reportedly activated by metabotropic glutamate receptors (Pastorino et al., 2000). Nevertheless, because at the high doses used in our study glutamate interacts with both ionotropic and metabotropic receptors (Sattler and Tymianski, 2001), it is likely that recruitment of both signaling pathways are involved in the deleterious effects of high glutamate on IGF-I signaling. Thus, more work is needed to elucidate this point. An important issue raised by these findings is whether attenuation of IGF-I prosurvival signaling unleashes proapoptotic pathways involved in glutamate excitotoxicity (Sattler and Tymianski, 2001). Indeed, inhibition of Akt activity; i.e., the prototype route of IGF-I prosurvival signaling, is sufficient to trigger neuronal death (Dudek et al., 1997). Therefore, homeostasis of neuron survival may include a tonic signaling mediated by activated Akt and possibly other prosurvival pathways. Under pathological circumstances, such as high glutamate, disruption of these tonic prosurvival pathways, which are intimately linked to death pathways (Datta et al., 1997), will tip the balance toward death processes. This death-triggering process may very well coexist with glutamate-induced intracellular Ca2⫹ rises (Fig. 6), i.e., PKC stimulates intracellular Ca2⫹ levels (Saitoh et al., 2001), and PKC is required for NMDA activation of NO synthase (Marin et al., 1992), a known effector of calcium neurotoxicity induced by glutamate (Sattler and Tymianski, 2000). Alternative cell-death signaling in NMDA-dependent excitotoxicity may predominate in specific types of excitotoxic damage. Thus, we suggest that activation of PKC by glutamate is a branching point toward Ca2⫹ excitotoxicity and proapoptotic signaling by interference with prosurvival signaling (routes 1 and 2 of Fig. 6). Our proposal agrees with previous findings indicating that PKC is needed for glutamate-induced changes in intracellular Ca2⫹ homeostasis and neuronal death (Favaron et al., 1990) and explains that inhibition of PKC is sufficient to abrogate glutamate-induced neuronal death. In our in vitro studies we employed cerebellar cells to obtain a relatively enriched neuronal population, but in vivo, the cerebellum is not usually affected by excitotoxic insults. Therefore, we analyzed the interaction between glutamate and IGF-I signaling in vivo in brain areas affected by this type of insult. As in cultured cerebellar cells, excess glutamate antagonizes IGF-I induced increases in pAkt in the hippocampus and phosphorylates IRS-1 in Ser307 residues, effects blocked by Ro320432. Additional support to an in vivo significance of this novel pathway involved in glutamate excitotoxicity in cultured cerebellar cells is the observation that in rats submitted to transient cerebral ischemia administration of Ro320432 diminished infarct size. Diverse in vivo observations such as that levels of IGF-I after ischemic lesions change (Schwab et al., 1997;

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O’Donnell et al., 2002) or that IGF-I is neuroprotective in animal models of ischemia (Johnston et al., 1996) indirectly involve IGF-I in neurotoxic pathways. However, detailed in vivo studies are required to determine whether modulation of PKC activity is a novel therapeutic target in excitotoxic injury. This possibility is particularly attractive in view of the existence of orally active PKC inhibitors such as Ro320432 (Birchall et al., 1994). Should this or equivalent compounds be able to circumvent the blood– brain barrier, its application appears feasible. Furthermore, PKC inhibitors may turn out to be therapeutic adjuvants to neuroprotective compounds such as IGF-I by reducing loss of sensitivity to neuroprotective input.

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and IGF-I signaling we routinely employed 500 ␮M glutamate and 100 nM IGF-I because the most robust responses were obtained with these high doses. Excitotoxic damage after 72 h was evaluated using propidium iodide (2 ␮g/ml) as a marker of cell death. Cultures were examined by fluoroscence microscopy (Zeiss, Germany) 30 min later. Cells within randomly located microscope fields were counted. At least 300 cells were scored per dish. A minimum of four independent experiments were done in duplicate dishes. When analyzing the effects of glutamate on IGF-I signaling, cultures were exposed to glutamate together with IGF-I for 3 min, whereas, when analyzing Akt phosphorylation, cells were stimulated 30 min (see Results). In vivo experiments

Experimental methods Animals and materials Wistar rats from our in-bred colony were kept following European Union guideline 86/609/EEC. IGF-I was from GroPep (Australia). Glutamate, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDCA, an inhibitor of glutamate uptake), forskolin, TPA (phorbol-14-myristate-13-acetate), okadaic acid, BAPTA-AM, and cyclosporin were from Sigma (St. Louis, MO, USA). Tautomycin, U73122, H89, bisindolylmaleimide I, Ro-320432, PP2, SB203580, KN-93 and K-252a, LY94002 and wortmannin, and PD98059 were from Calbiochem (USA). The doses used for the different drugs were always supraoptimal. To avoid interference with other pathways we used well-established doses. AntiphosphoTyr antibody (PY20), anti-pSer729 protein kinase C (PKC)␧, and anti-pSer307 insulin receptor substrate (IRS)-1 were from Upstate Biotechnology (USA). Anti-pSer473-Akt and pThr183/Thr185-Jun Kinase (JNK) were from New England Biolabs (USA). Anti-Akt was from Santa Cruz Laboratories (USA). A pan-PKC antibody that recognizes the 80-kDa polypeptide of PKC was from Sigma. Antibodies specific for the IGF-I receptor, IRS-1, and phosphatidylinositol kinase (PI3K)p85 were developed as described (Trejo and Pons, 2001). Neuronal cultures Cerebellar granule cultures from 7-day-old rats were prepared as described (Gonzalez de la Vega et al., 2001). Cells were grown (2 ⫻ 106 cells/dish) on polystyrene dishes with Neurobasal ⫹ B27 (Gibco, USA), glutamine, and 25 mM KCl. Under these conditions, ⬃95% of the cells are neurons (␤3-tubulin-positive). In the day of the experiment, medium was replaced with Neurobasal ⫹ 25 mM KCl. Two hours later IGF-I (1–100 nM) and/or glutamate (5 ␮M to 1 mM) were added while inhibitory drugs were given 45 min before treatments. Although glutamate doses below 100 ␮M were not cytotoxic, all doses tested of IGF-I were neurotrophic. However, to analyze interactions between glutamate

Deeply anesthesized male rats (250 –300 g) were injected into the cerebral lateral ventricle (icv) following the stereotaxic coordinates: 1.4 mm lateral, 4 mm ventral, and 0.8 mm from Bregma (Paxinos and Watson, 1982) with glutamate (10 mM), PDCA (15 mM), and IGF-I (100 ␮M) or IGF-I alone in a total volume of 5 ␮l. PDCA, a glutamate reuptake inhibitor, was used to maintain high extracellular levels of glutamate. Controls received saline. Another group of animals were injected icv with Ro320432 (100 ␮M) 45 min prior to treatment. To determine optimal time of stimulation, a time-course analysis of the effects of IGF-I on pAkt levels in the hippocampus was performed comparing pAkt levels at the injection site (ipsilateral) with those in the uninjected contralateral site to minimize the use of animals. Animals were sacrificed at the appropriate time points, and the hippocampus was dissected, snap frozen, and processed for Western blot. In a second series of experiments male rats were subjected to ischemic injury by occlusion of the medial cerebral artery and reperfusion 90 min later, as described (Dietrich, 1994). During occlusion, animals were implanted subcutaneously with an osmotic minipump (Alzet 1007D) connected to an icv cannula stereotaxically placed 3 days before, as described (Carro et al., 2000). Pumps were filled with either Ro324032 (100 mM) or saline. A week later animals were sacrificed, their brain perfused with 4% paraformaldehyde, and 50-␮m sections cut along the entire rostrocaudal extent of the lesion, as described (Trejo and Pons, 2001). Evaluation of infarct size was performed following procedures described elsewhere (Osborne et al., 1987; Wexler et al., 2002). Briefly, sections were stained with hematoxylin-eosin and coded, photographs of brain sections were taken every 1.5 mm under an stereoscope, and lesion volume was calculated by a blind observer using image analysis software (IAS). At least five animals were treated per group. Immunoassays Tissue and cell samples were lysed and either immunoprecipitated or directly subjected to Western blotting as

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described (Pons and Torres-Aleman, 2000). To normalize for protein load, membranes were reblotted with either the same antibody used for immunoprecipitation or with an appropriate control antibody (see Results). Levels of the protein under study were expressed relative to protein load in each lane. Densitometric analysis was performed using 3Dimage software (Spain). Results shown are from at least four independent experiments. Acknowledgments The authors are grateful to J. Sancho and F. Lozano for their expert help and to C. Bailon for assistance in artwork. This work was supported by grants from FIS (01/1188) and DGI (SAF 2001-1722). References Aguirre, V., Werner, E.D., Giraud, J., Lee, Y.H., Shoelson, S.E., White, M.F., 2002. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J. Biol. Chem. 277, 1531–1537. Bandrowski, A.E., Ashe, J.H., Crawford, C.A., 2001. Tetanic stimulation and metabotropic glutamate receptor agonists modify synaptic responses and protein kinase activity in rat auditory cortex. Brain Res. 894, 218 –232. Beal, M.F., 1992. Mechanisms of excitotoxicity in neurologic diseases. FASEB J. 6, 3338 –3344. Beaver, C.J., Ji, Q., Fischer, Q.S., Daw, N.W., 2001. Cyclic AMP-dependent protein kinase mediates ocular dominance shifts in cat visual cortex. Nat. Neurosci. 4, 159 –163. Birchall, A.M., Bishop, J., Bradshaw, D., Cline, A., Coffey, J., Elliott, L.H., Gibson, V.M., Greenham, A., Hallam, T.J., Harris, W., 1994. Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation. J. Pharmacol. Exp. Ther. 268, 922–929. Bulleit, R.F., Cui, H., 1998. Methylmercury antagonizes the survivalpromoting activity of insulin-like growth factor on developing cerebellar granule neurons. Toxicol. Appl. Pharmacol. 153, 161–168. Busiguina, S., Fernandez, A.M., Barrios, V., Clark, R., Tolbert, D.L., Berciano, J., Torres-Aleman, I., 2000. Neurodegeneration is associated to changes in serum insulin-like growth factors. Neurobiol. Dis. 7, 657– 665. Calabresi, P., Saulle, E., Marfia, G.A., Centonze, D., Mulloy, R., Picconi, B., Hipskind, R.A., Conquet, F., Bernardi, G., 2001. Activation of metabotropic glutamate receptor subtype 1/protein kinase C/mitogenactivated protein kinase pathway is required for postischemic long-term potentiation in the striatum. Mol. Pharmacol. 60, 808 – 815. Carro, E., Nunez, A., Busiguina, S., Torres-Aleman, I., 2000. Circulating insulin-like growth factor I mediates effects of exercise on the brain. J. Neurosci. 20, 2926 –2933. Carro, E., Trejo, J.L., Gomez-Isla, T., LeRoith, D., Torres-Aleman, I., 2002. Serum insulin-like growth factor I regulates brain amyloid-beta levels. Nat. Med. 8, 1390 –1397. Cenni, V., Doppler, H., Sonnenburg, E.D., Maraldi, N., Newton, A.C., Toker, A., 2002. Regulation of novel protein kinase C epsilon by phosphorylation. Biochem. J. 363, 537–545. Chalecka-Franaszek, E., Chuang, D.M., 1999. Lithium activates the serine/ threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc. Natl. Acad. Sci. USA 96, 8745– 8750. D’Ercole, A.J., Ye, P., Calikoglu, A.S., Gutierrez-Ospina, G., 1996. The role of the insulin-like growth factors in the central nervous system. Mol. Neurobiol. 13, 227–255.

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