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Glycogen Synthase Kinase-3β Activity Is Critical for Neuronal Death Caused by Inhibiting Phosphatidylinositol 3-Kinase or Akt but Not for Death Caused by Nerve Growth Factor Withdrawal*

  • Robert J. Crowder
    Footnotes
    Affiliations
    Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
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  • Robert S. Freeman
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-4893; Fax: 716-273-2652
    Affiliations
    Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
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  • Author Footnotes
    * This work was supported in part by a generous contribution from the Paul Stark Endowment at the University of Rochester and by National Institutes of Health Grant NS34400.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by Predoctoral Training Program Grant AG00107 from the National Institutes of Health.
Open AccessPublished:November 03, 2000DOI:https://doi.org/10.1074/jbc.M006160200
      Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3β (GSK-3β), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3β is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3β and inhibition of its protein kinase activity, indicating that GSK-3β is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3β inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3β, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3β reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3β caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3β, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3β function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3β-independent pathways.
      NGF
      nerve growth factor
      PI
      phosphatidylinositol
      GSK-3β
      glycogen synthase kinase-3β
      GBP
      Xenopus GSK-3-binding protein
      PBS
      phosphate-buffered saline
      GSK-3βKM
      kinase-deficient glycogen synthase kinase-3β mutant
      TBST
      Tris-buffered saline containing Tween 20
      IRS-1
      insulin receptor substrate 1
      The survival of developing neurons and perhaps most cells requires extracellular cues to actively prevent programmed cell death. In the nervous system such cues are provided in part by neurotrophic factors, such as the nerve growth factor (NGF),1 that bind to tyrosine protein kinase receptors and activate intracellular signaling pathways (
      • Kaplan D.R.
      • Miller F.D.
      ). One such pathway initiated by NGF involves the generation of 3′-phosphorylated phosphoinositides via the activation of phosphatidylinositol (PI) 3-kinase (
      • Carter A.N.
      • Downes C.P.
      ,
      • Raffioni S.
      • Bradshaw R.A.
      ,
      • Soltoff S.P.
      • Rabin S.L.
      • Cantley L.C.
      • Kaplan D.R.
      ). This pathway has recently emerged as an important mechanism by which NGF and other neurotrophins promote cell survival (
      • Kaplan D.R.
      • Miller F.D.
      ). The primary effector of PI 3-kinase for cell survival is the Akt serine/threonine protein kinase, which is activated in cells stimulated with NGF in a PI 3-kinase-dependent manner (
      • Andjelkovich M.
      • Suidan H.S.
      • Meier R.
      • Frech M.
      • Alessi D.R.
      • Hemmings B.A.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ). Importantly, inhibitors of PI 3-kinase and Akt block the survival-promoting effects of NGF and other neurotrophic factors, and activated forms of PI 3-kinase and Akt promote neuronal survival in the absence of external survival factors (
      • Crowder R.J.
      • Freeman R.S.
      ,
      • D'Mello S.R.
      • Borodezt K.
      • Soltoff S.P.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Miller T.M.
      • Tansey M.G.
      • Johnson Jr., E.M.
      • Creedon D.J.
      ,
      • Bartlett S.E.
      • Reynolds A.J.
      • Weible M.
      • Heydon K.
      • Hendry I.A.
      ,
      • Hetman M.
      • Kanning K.
      • Cavanaugh J.E.
      • Xia Z.
      ,
      • Mazzoni I.E.
      • Said F.A.
      • Aloyz R.
      • Miller F.D.
      • Kaplan D.
      ,
      • Vaillant A.R.
      • Mazzoni I.
      • Tudan C.
      • Boudreau M.
      • Kaplan D.R.
      • Miller F.D.
      ).
      Despite the significance of the PI 3-kinase/Akt pathway for neuronal survival, the mechanisms that lie downstream of Akt and mediate survival remain obscure. Recent findings in neurons and nonneuronal cells suggest that Akt may function by inhibiting death-promoting proteins (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). For example, Akt can phosphorylate the pro-apoptotic Bcl-2 family member, BAD, as well as members of the Forkhead family of transcriptional regulators. Phosphorylation of BAD or the Forkhead transcription factor FKHRL1 by Akt suppresses the death-promoting activity of these and other proteins (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), suggesting at least two ways that Akt can contribute to cell survival.
      Glycogen synthase kinase-3β (GSK-3β) was the first Akt substrate shown to be inhibited upon phosphorylation by Akt (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). By expressing a catalytically inactive form of GSK-3β in Rat-1 fibroblasts and in undifferentiated pheochromocytoma PC12 cells, Pap and Cooper (
      • Pap M.
      • Cooper G.M.
      ) showed that GSK-3β function is required for cell death that is caused by the inhibition of PI 3-kinase. Stimulation of PC12 cells with NGF caused a PI 3-kinase-dependent reduction in GSK-3β activity, raising the possibility that inhibition of GSK-3β may be a critical component of NGF-dependent survival in PC12 cells. More recently, inhibitors of GSK-3β were shown to reduce cell death in rat cortical neurons when death was initiated by the PI 3-kinase inhibitor LY294002, serum deprivation, or serum deprivation combined with exposure to an N-methyl-d-aspartate receptor antagonist (
      • Hetman M.
      • Cavanaugh J.E.
      • Kimelman D.
      • Xia Z.G.
      ). Although these studies suggest a role for GSK-3β in neuronal death, the importance of regulating GSK-3β activity for the survival of neurons by their physiological neurotrophic factor has not been examined.
      In this study, we have used NGF-dependent rat sympathetic neurons to compare the role of GSK-3β in NGF withdrawal-induced death with its role in death caused by inhibiting PI 3-kinase or Akt. Our results indicate that GSK-3β is a target of PI 3-kinase and Akt in sympathetic neurons but that inhibition of GSK-3β activity is not sufficient to block apoptosis caused by NGF withdrawal.

      DISCUSSION

      Inhibitors of GSK-3β activity have recently been shown to reduce cell death caused by PI 3-kinase inhibition or serum withdrawal in PC12 cells and cortical neurons (
      • Pap M.
      • Cooper G.M.
      ,
      • Hetman M.
      • Cavanaugh J.E.
      • Kimelman D.
      • Xia Z.G.
      ). However, the importance of GSK-3β for cell death in neurons deprived of their physiological survival factor has not been tested previously. Sympathetic neurons from neonatal rat superior cervical ganglia, which require NGF for survival in vivo and in vitro, provide a powerful model for studying neurotrophic factor dependence (
      • Deshmukh M.
      • Johnson E.M.
      ). We used these cells to compare the role of GSK-3β in neuronal death caused by inhibition of the PI 3-kinase/Akt pathway with its role in death caused by NGF withdrawal. Our results corroborate previous findings that GSK-3β is a target of the PI 3-kinase pathway in primary neurons and that inhibition of GSK-3β can protect neurons from death induced by PI 3-kinase inhibitors. In addition, we demonstrate that inhibition of GSK-3β reduces neuronal death caused by the inhibition of Akt, suggesting that endogenous Akt acts to inhibit GSK-3β in neurons. Finally, our results provide evidence that inhibiting GSK-3β is not sufficient to block cell death after NGF withdrawal.
      Stimulation of sympathetic neurons with NGF caused a PI 3-kinase-dependent increase in GSK-3β phosphorylation on serine 9 and a parallel decrease in GSK-3β kinase activity. Because Akt can phosphorylate serine 9 and inactivate GSK-3β after insulin stimulation (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ), it is probable that the NGF-induced inhibition of GSK-3β activity seen here is mediated by a similar Akt-dependent mechanism. Nonetheless, it remains possible that regulation of GSK-3β by NGF may involve other PI 3-kinase effectors (
      • Kobayashi T.
      • Cohen P.
      ). Our observation that both Frat1 and kinase-defective GSK-3β provide protection against AH-Akt-induced death suggests that GSK-3β is at least partly regulated by Akt in response to NGF treatment.
      A role for GSK-3β activity in neuronal death has recently been demonstrated in cortical neurons. Withdrawal of serum from cortical neurons caused an increase in GSK-3β activity that preceded the onset of cell death and that was reversed by the addition of brain-derived neurotrophic factor (
      • Hetman M.
      • Cavanaugh J.E.
      • Kimelman D.
      • Xia Z.G.
      ). When cortical neurons were transiently transfected with expression vectors for GBP or kinase-deficient GSK-3β, apoptosis caused by serum deprivation or by PI 3-kinase inhibition was significantly reduced. Overexpression of wild-type GSK-3β in cortical neurons caused a modest increase in neuronal death. These data are consistent with a role for GSK-3β in the death of neurons deprived of survival factors. In agreement with such a role, we found that overexpression of GSK-3β in sympathetic neurons could partially override the survival signals initiated by NGF, resulting in a 20–30% increase in cell death. However, none of the GSK-3β inhibitors that we tested (Frat1, GBP, or GSK-3βKM) was sufficient to prevent the death of NGF-deprived sympathetic neurons. This lack of protection was not simply a consequence of inadequate expression of these proteins as both Frat1 and GSK-3βKM were expressed sufficiently in neurons to inhibit death caused by blocking Akt function.
      Why do GSK-3β inhibitors prevent death of serum-deprived cortical neurons but not NGF-deprived sympathetic neurons? GSK-3β activity is regulated not only by phosphorylation on serine residues but also through its association with proteins, such as Frat1, Axin, and Dishevelled (
      • Li L.
      • Yuan H.
      • Weaver C.D.
      • Mao J.
      • Farr G.H.
      • Sussman D.J.
      • Jonkers J.
      • Kimelman D.
      • Wu D.
      ). GSK-3β-binding proteins appear to inhibit GSK-3β kinase activity toward certain substrates but not other substrates (
      • Thomas G.M.
      • Frame S.
      • Goedert M.
      • Nathke I.
      • Polakis P.
      • Cohen P.
      ), and recent reports suggest that kinase-deficient GSK-3β mutants might not block all downstream pathways regulated by GSK-3 (
      • Yuan H.
      • Mao J.
      • Li L.
      • Wu D.
      ,
      • Smalley M.J.
      • Sara E.
      • Paterson H.
      • Naylor S.
      • Cook D.
      • Jayatilake H.
      • Fryer L.G.
      • Hutchinson L.
      • Fry M.J.
      • Dale T.C.
      ). Consequently, the precise effects of overexpressing GSK-3-binding proteins and kinase-deficient GSK-3β mutants are difficult to ascertain and may vary in different cell types. In addition, different cells may vary in their dependence on the function of various GSK-3β substrates, such as glycogen synthase, eIF2B, Tau, and β-catenin (
      • Welsh G.I.
      • Wilson C.
      • Proud C.G.
      ). For example, destabilization of β-catenin, which occurs as a consequence of its phosphorylation by GSK-3β (
      • Yost C.
      • Torres M.
      • Miller J.R.
      • Huang E.
      • Kimelman D.
      • Moon R.T.
      ), has been reported to enhance cell death in hippocampal neurons (
      • Zhang Z.H.
      • Hartmann H.
      • Do V.M.
      • Abramowski D.
      • Sturchlerpierrat C.
      • Staufenbiel M.
      • Sommer B.
      • Vandewetering M.
      • Clevers H.
      • Saftig P.
      • Destrooper B.
      • He X.
      • Yankner B.A.
      ). This observation suggests that stabilization of β-catenin through down-regulation of GSK-3β activity could be one mechanism by which neurotrophins promote survival in neurons. However, expression of a stabilized form of β-catenin failed to protect cortical neurons from death caused by serum withdrawal or inhibition of PI 3-kinase, indicating that at least in some cases GSK-3β-mediated destabilization of β-catenin is not necessary for neuronal death (
      • Hetman M.
      • Cavanaugh J.E.
      • Kimelman D.
      • Xia Z.G.
      ). GSK-3-mediated phosphorylation of insulin receptor substrate 1 (IRS-1) has been shown to inhibit insulin signaling in nonneuronal cells (
      • Eldar-Finkelman H.
      • Krebs E.G.
      ), and by analogy it could play a similar role in negatively regulating neurotrophin-derived survival signals in neurons. A report that IRS-1 can mediate the brain-derived neurotrophic factor-dependent activation of PI 3-kinase in cortical neurons is consistent with this possibility (
      • Yamada M.
      • Ohnishi H.
      • Sano S.
      • Nakatani A.
      • Ikeuchi T.
      • Hatanaka H.
      ). However, a role for IRS-1 in NGF signaling has not been reported. Whereas IRS-1 may be regulated by GSK-3β in brain-derived neurotrophic factor-responsive cortical neurons, it seems unlikely to be an important GSK-3β target in NGF-dependent neurons.
      Although inhibitors of GSK-3β failed to prevent death caused by NGF withdrawal, they did protect neurons from death caused by the inhibition of PI 3-kinase and Akt. One explanation for this apparent discrepancy could be that NGF withdrawal leads to higher levels of GSK-3β activity than does PI 3-kinase or Akt inhibition. In this case, the GSK-3β inhibitors might not be expressed sufficiently to inhibit the potentially higher level of GSK-3β activity in NGF-deprived neurons. This scenario seems unlikely because the level of GSK-3β kinase activity detected in PI 3-kinase inhibitor-treated neurons was not different from that of NGF-deprived neurons (Fig. 1). Moreover, treatment with LiCl at concentrations at and above those known to inhibit GSK-3β kinase activity (
      • Klein P.S.
      • Melton D.A.
      ,
      • Stambolic V.
      • Ruel L.
      • Woodgett J.R.
      ,
      • Hong M.
      • Chen D.C.R.
      • Klein P.S.
      • Lee V.M.-Y.
      ) failed to provide protection from NGF withdrawal. Our results favor a model in which NGF withdrawal leads to the activation of cell death pathways in addition to those activated by PI 3-kinase inhibitors. Although pro-apoptotic proteins, such as BAD, Caspase-9 (human), and FKHRL1 can be negatively regulated by Akt (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), they can also be regulated by additional pathways (
      • Harada H.
      • Becknell B.
      • Wilm M.
      • Mann M.
      • Huang L.S.
      • Taylor S.S.
      • Scott J.D.
      • Korsmeyer S.J.
      ,
      • Fujita E.
      • Jinbo A.
      • Matuzaki H.
      • Konishi H.
      • Kikkawa U.
      • Momoi T.
      ). Therefore, removal of NGF might result in their activation to a level beyond that achieved by inhibition of PI 3-kinase and Akt alone. In addition, other pro-apoptotic mechanisms are up-regulated after NGF withdrawal including c-Jun NH2-terminal kinase activation, translocation of Bax to mitochondria, and release of cytochrome c from mitochondria (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ,
      • Virdee K.
      • Bannister A.J.
      • Hunt S.P.
      • Tolkovsky A.M.
      ,
      • Eilers A.
      • Whitfield J.
      • Babij C.
      • Rubin L.L.
      • Ham J.
      ,
      • Putcha G.V.
      • Deshmukh M.
      • Johnson E.M.
      ,
      • Deshmukh M.
      • Johnson E.M.
      ,
      • Neame S.J.
      • Rubin L.L.
      • Philpott K.L.
      ). Activation of one or more of these pathways after NGF withdrawal could result in cell death that is independent of GSK-3β activity. Finally, an alternative explanation for these results is that NGF withdrawal, but not PI 3-kinase/Akt inhibition, could conceivably activate GSK-3β functions that are not targeted by the inhibitors used in this study.
      NGF activates proteins in addition to PI 3-kinase and Akt that can function to promote cell survival. For example, the transcription factors NF-κB (
      • Wood J.N.
      ,
      • Carter B.D.
      • Kaltschmidt C.
      • Kaltschmidt B.
      • Offenhauser N.
      • Bohm M.
      • Baeuerle P.A.
      • Barde Y.A.
      ) and cAMP-response element-binding protein (
      • Ginty D.D.
      • Bonni A.
      • Greenberg M.E.
      ) are activated by NGF and have recently been shown to contribute to NGF-dependent survival in neurons (
      • Maggirwar S.B.
      • Sarmiere P.D.
      • Dewhurst S.
      • Freeman R.S.
      ,
      • Riccio A.
      • Ahn S.
      • Davenport C.M.
      • Blendy J.A.
      • Ginty D.D.
      ). Decreased activity of these transcription factors after NGF withdrawal might lead to cell death even in the presence of GSK-3β inhibitors, possibly via decreased expression of anti-apoptotic Bcl-2 family members (
      • Riccio A.
      • Ahn S.
      • Davenport C.M.
      • Blendy J.A.
      • Ginty D.D.
      ,
      • Tamatani M.
      • Che Y.H.
      • Matsuzaki H.
      • Ogawa S.
      • Okado H.
      • Miyake S.
      • Mizuno
      • Tohyama M.
      ,
      • Grumont R.J.
      • Rourke I.J.
      • Gerondakis S.
      ,
      • Zong W.X.
      • Edelstein L.C.
      • Chen C.
      • Bash J.
      • Gelinas C.
      ).
      Although our results indicate that GSK-3β inhibition by itself is not sufficient to maintain the survival of NGF-deprived neurons, the inhibition of GSK-3β activity is clearly an important aspect of the survival mechanisms mediated by PI 3-kinase and Akt. Exactly how GSK-3β activity influences the many other effectors of cell death and survival remains to be determined.

      Acknowledgments

      We thank Drs. J. Woodgett, P. Saint-Jeannet, and X. He for providing GSK-3 expression plasmids. We thank Dr. D. Kimelman for the GBP clone, Dr. D. Wu for the Frat1 and FratN expression vectors, and Dr. E. M. Johnson for the Bax expression vector. We also thank Pat Sarmiere for help with plasmid construction and Daphne Hasbani and Leah Larocque for the preparation of tissue culture reagents.

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