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Neuroprotection by Brain-derived Neurotrophic Factor Is Mediated by Extracellular Signal-regulated Kinase and Phosphatidylinositol 3-Kinase*

  • Michal Hetman
    Footnotes
    Affiliations
    Toxicology Program, Department of Environmental Health, the Graduate Program in Neurobiology and Behavior, the Graduate Program in Molecular and Cell Biology

    Department of Pharmacology, University of Washington, Seattle, Washington 98195-7234
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  • Kevin Kanning
    Affiliations
    Toxicology Program, Department of Environmental Health, the Graduate Program in Neurobiology and Behavior, the Graduate Program in Molecular and Cell Biology
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  • Jane E. Cavanaugh
    Footnotes
    Affiliations
    Toxicology Program, Department of Environmental Health, the Graduate Program in Neurobiology and Behavior, the Graduate Program in Molecular and Cell Biology

    Department of Pharmacology, University of Washington, Seattle, Washington 98195-7234
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  • Zhengui Xia
    Correspondence
    To whom correspondence should be addressed: Dept. of Environmental Health, Box 357234, University of Washington, HSB, Room F561C, Seattle, WA 98195. Tel: 206-616-9433; Fax: 206-685-3990,
    Affiliations
    Toxicology Program, Department of Environmental Health, the Graduate Program in Neurobiology and Behavior, the Graduate Program in Molecular and Cell Biology
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Sheldon Murphy Endowment Fund, by pilot grants from the American Cancer Society Institutional Cancer Research Grant at University of Washington and the Nathan Shock Center in the Basic Biology of Aging and the Alzheimer's Disease Research Center at University of Washington, and by Grant NS37359 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (to Z. X.).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.
    ¶ Completed part of this work during the tenure of a fellowship award from the American Heart Association, Washington Affiliate.
    ‖ Supported by National Institutes of Health Post-doctoral Training Grant “Genetic Approaches to Aging” (2 T32 AG00057-21).
Open AccessPublished:August 06, 1999DOI:https://doi.org/10.1074/jbc.274.32.22569
      Apoptosis is a form of programmed cell death that plays a pivotal role during development and in the homeostasis of the adult nervous systems. However, mechanisms that regulate neuronal apoptosis are not well defined. Here, we report that brain-derived neurotrophic factor (BDNF) protects cortical neurons against apoptosis induced by camptothecin or serum deprivation and activates the extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Using pharmacological agents and transient transfection with dominant interfering or constitutive active components of the ERK or the PI 3-kinase pathway, we demonstrate that the ERK pathway plays a major role in BDNF neuroprotection against camptothecin. Furthermore, ERK is activated in cortical neurons during camptothecin-induced apoptosis, and inhibition of ERK increases apoptosis. In contrast, the PI 3-kinase pathway is the dominant survival mechanism for serum-dependent survival under normal culture conditions and for BDNF protection against serum withdrawal. These results suggest that the ERK pathway is one of several neuroprotective mechanisms that are activated by stress to counteract death signals in central nervous system neurons. Furthermore, the relative contribution of the ERK and PI 3-kinase pathways to neuronal survival may depend on the type of cellular injury.
      There is considerable interest in elucidating the mechanisms for apoptosis in central nervous system (CNS)
      The abbreviations used are: CNS, central nervous system; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BME, basal medium Eagle; ERK, extracellular signal-regulated kinase; PI 3-kinase, phosphatidylinositol 3-kinase; NGF, nerve growth factor; HA, hemagglutinin; DIV, days in in vitroculture; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MKK1, MAP kinase-kinase 1; MAP-2, microtubule-associated protein 2; SCG, superior cervical ganglion
      1The abbreviations used are: CNS, central nervous system; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BME, basal medium Eagle; ERK, extracellular signal-regulated kinase; PI 3-kinase, phosphatidylinositol 3-kinase; NGF, nerve growth factor; HA, hemagglutinin; DIV, days in in vitroculture; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MKK1, MAP kinase-kinase 1; MAP-2, microtubule-associated protein 2; SCG, superior cervical ganglion
      neurons because apoptosis plays an important role during neuronal development (
      • Raff M.C.
      • Barres B.A.
      • Burne J.
      • Coles H.S.
      • Ishizaki Y.
      • Jacobson M.D.
      ,
      • Oppenheim R.W.
      ). Furthermore, defects in the control of apoptosis may contribute to several pathologies in the CNS including stroke, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Alzheimer's disease (
      • Raff M.C.
      • Barres B.A.
      • Burne J.
      • Coles H.S.
      • Ishizaki Y.
      • Jacobson M.D.
      ,
      • Stefanis L.
      • Burke R.E.
      • Greene L.A.
      ). However, most mechanistic studies of apoptosis have been limited to proliferating, non-neuronal cells or neurons derived from the peripheral nervous system, including superior cervical ganglion (SCG) or dorsal root ganglion neurons. Although mature neurons and proliferating non-neuronal cells may share some apoptotic mechanisms, mature CNS neurons do not divide. Consequently, there may be significant differences in the mechanisms for apoptosis in CNS neurons and dividing cells. For example, cytosine arabinoside, a DNA synthesis inhibitor, triggers apoptosis in both dividing cells and post-mitotic neurons (
      • Martin D.P.
      • Wallace T.L.
      • Johnson Jr., E.M.
      ). However, cycloheximide, a protein synthesis inhibitor, potentiates apoptosis in many dividing cells but inhibits apoptosis in post-mitotic neurons (
      • Raff M.C.
      • Barres B.A.
      • Burne J.
      • Coles H.S.
      • Ishizaki Y.
      • Jacobson M.D.
      ,
      • Stefanis L.
      • Burke R.E.
      • Greene L.A.
      ,
      • Koh J.Y.
      • Wie M.B.
      • Gwag B.J.
      • Sensi S.L.
      • Canzoniero L.M.
      • Demaro J.
      • Csernansky C.
      • Choi D.W.
      ,
      • Kharlamov E.
      • Cagnoli C.M.
      • Atabay C.
      • Ikonomovic S.
      • Grayson D.R.
      • Manev H.
      ,
      • Lindenboim L.
      • Haviv R.
      • Stein R.
      ,
      • Saura J.
      • MacGibbon G.
      • Dragunow M.
      ). Because the biochemical and regulatory properties of post-mitotic CNS neurons are distinct from those of peripheral nervous system neurons and dividing non-neuronal cells, it is crucial to define apoptotic mechanisms specific to CNS neurons; this may ultimately lead to the identification of drug targets for modulation of neuronal apoptosis in the CNS and the development of clinical strategies for treatment of neurodegenerative disorders.
      The regulatory mechanisms that control neuronal survival and apoptosis are just beginning to be defined (
      • Estus S.
      ,
      • Green D.R.
      • Reed J.C.
      ). Many growth factors and neurotrophins can promote neuronal survival, including insulin, insulin-like growth factor-1, BDNF, nerve-growth factor (NGF), and neurotrophins 3 and 4/5 (
      • Levi-Montalcini R.
      • Booker B.
      ,
      • Greene L.A.
      ,
      • Barde Y.-A.
      ,
      • Datta S.R.
      • Greenberg M.E.
      ). These factors can also activate several intracellular signaling transduction systems including the ERK and the PI 3-kinase pathways (
      • Castellino A.M.
      • Chao M.V.
      ,
      • Segal R.A.
      • Greenberg M.E.
      ). Activation of the PI 3-kinase pathway is required for NGF-mediated survival of PC12 cells and SCG neurons (
      • Yao R.
      • Cooper G.M.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ), for insulin-like growth factor-1-mediated survival of cerebellar granule neurons, oligodendrocytes, and PC12 cells (
      • D'Mello S.R.
      • Borodezt K.
      • Soltoff S.P.
      ,
      • Miller T.M.
      • Tansey M.G.
      • Johnson E.M.
      • Creedon D.J.
      ,
      • Vemuri G.S.
      • McMorris F.A.
      ,
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ), and for membrane depolarization-mediated survival of cerebellar granule neurons (
      • Miller T.M.
      • Tansey M.G.
      • Johnson E.M.
      • Creedon D.J.
      ). Furthermore, protein kinase Akt (also known as PKB or RAC) may mediate cellular survival because of activation of PI 3-kinase in cerebellar granule neurons and other non-neuronal cells (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.J.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Kauffmann-Zeh A.
      • RodriguezViciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ). Collectively, these data have established the PI 3-kinase pathway as a major neuroprotective mechanism (
      • Datta S.R.
      • Greenberg M.E.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ).
      Although activation of the ERK signaling pathway protects various non-neuronal cell lines against apoptosis (
      • Carter S.
      • Auer K.L.
      • Reardon D.B.
      • Birrer M.
      • Fisher P.B.
      • Valerie K.
      • SchmidtUllrich R.
      • Mikkelsen R.
      • Dent P.
      ,
      • Gardner A.M.
      • Johnson G.L.
      ,
      • Guyton K.Z.
      • Liu Y.
      • Gorospe M.
      • Xu Q.
      • Holbrook N.J.
      ), its role for promoting the survival of neurons is still controversial. Activation of ERK promotes PC12 cell survival (
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ,
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ,
      • Yan C.Y.I.
      • Greene L.A.
      ). Furthermore, studies with pharmacological inhibitors suggest that ERK activation may mediate neuroprotection by BDNF in retinal ganglion cells and cerebellar neurons (
      • Meyer-Franke A.
      • Wilkinson G.A.
      • Kruttgen A.
      • Hu M.
      • Munro E.
      • Hanson M.G.
      • Reichardt L.F.
      • Barres B.A.
      ,
      • Skaper S.D.
      • Floreani M.
      • Negro A.
      • Facci L.
      • Giusti P.
      ), by NGF in sympathetic neurons (
      • Anderson C.N.G.
      • Tolkovsky A.M.
      ), and by the pituitary adenylate cyclase-activating polypeptide (PACAP-38) in cerebellar neurons (
      • Villalba M.
      • Journot L.
      ). However, other studies using inhibitors have suggested that ERK does not mediate the neuroprotection afforded by neurotrophins (NGF, BDNF, and insulin-like growth factor-1) or membrane depolarization in PC12, SCG, or cerebellar neurons (
      • Miller T.M.
      • Tansey M.G.
      • Johnson E.M.
      • Creedon D.J.
      ,
      • Virdee K.
      • Tolkovsky A.M.
      ,
      • Creedon D.J.
      • Johnson E.M.
      • Lawrence J.C.
      ,
      • Gunn Moore F.J.
      • Williams A.G.
      • Toms N.J.
      • Tavar'e J.M.
      ).
      The objective of this study was to evaluate the roles of the ERK and the PI 3-kinase pathways for BDNF neuroprotection of cortical neurons. Cortical neurons were chosen because the importance of ERK for neuroprotection in cortical neurons had not been examined and neurons in the cortex are frequently damaged during neurodegenerative diseases. Because different neuroprotective mechanisms may be activated to combat distinct types of cellular stress, we used two apoptotic paradigms: apoptosis induced by serum deprivation or by camptothecin treatment. Because the optimal growth and survival of neurons depend on the availability of growth factors and neurotrophic factors, serum withdrawal has been widely used as a model for CNS neuronal apoptosis induced by developmental cues (
      • D'Mello S.R.
      • Galli C.
      • Ciotti T.
      • Calissano P.
      ,
      • Koh J.-Y.
      • Gwag B.J.
      • Lobner D.
      • Choi D.W.
      ). Camptothecin is an inhibitor of DNA topoisomerase-1. It induces DNA strand breaks during replication as well as transcription, and it may inhibit transcription (
      • Morris E.J.
      • Geller H.M.
      ,
      • Rothenberg M.L.
      ). Camptothecin-induced apoptosis has been used as a model system to study neuronal apoptosis induced by DNA damage, which may contribute to several neurodegenerative diseases and aging-related neuron loss (
      • Morris E.J.
      • Geller H.M.
      ,
      • Park D.S.
      • Morris E.J.
      • Greene L.A.
      • Geller H.M.
      ,
      • Park D.S.
      • Morris E.J.
      • Padmanabhan J.
      • Shelanski M.L.
      • Geller H.M.
      • Greene L.A.
      ,
      • Park D.S.
      • Morris E.J.
      • Stefanis L.
      • Troy C.M.
      • Shelanski M.L.
      • Geller H.M.
      • Greene L.A.
      ). Our results suggest that the ERK signaling pathway plays a pivotal role in the BDNF protection against camptothecin, whereas the PI 3-kinase pathway is critical for BDNF protection against serum deprivation.

      DISCUSSION

      The objective of this study was to define the relative contribution of the ERK and the PI 3-kinase pathways to the protection of CNS neurons from different forms of cellular stress. Because CNS neurons have unique cellular and biochemical features that distinguish them from non-CNS neurons, it is important to identify apoptotic mechanisms for CNS neurons. Furthermore, it is likely that several anti-apoptotic mechanisms are employed for the protection of CNS neurons with specificity for the apoptotic signal and type of neurons. One of the obstacles impeding the study of apoptosis in the CNS has been the difficulty in transfecting post-mitotic CNS neurons with high efficiency and low toxicity. The modified calcium phosphate transfection method used in this study allowed us to transfect neurons in culture with specific genes of interest, e.g.constitutively active MKK1 or PI 3-kinase.
      The data presented here indicate that camptothecin treatment or serum deprivation, two distinct forms of stress, induce cell death in cortical neurons with phenotypes characteristic of apoptosis. BDNF protected cortical neurons from both stimuli, albeit by different mechanisms. Both the ERK and the PI 3-kinase signal transduction systems were activated by BDNF in cortical neurons. Blocking the ERK pathway using the pharmacological agent PD98059 inhibited BDNF neuroprotection against camptothecin but not serum deprivation. Furthermore, selective and constitutive activation of ERK by transient expression of a constitutive active MKK1 protected cortical neurons from apoptosis induced by camptothecin but not serum deprivation. Although drugs and transient expression experiments have the potential to exhibit nonspecific effects, both approaches gave similar results in our study, strengthening our conclusion that ERK is important for BDNF protection against camptothecin. In contrast, inhibition of the PI 3-kinase pathway by LY294002 was very effective in reversing the neuroprotection of BDNF against serum deprivation but not camptothecin treatment. Expression of a constitutive active PI 3-kinase protected cortical neurons from apoptosis induced by serum deprivation but not camptothecin treatment. Moreover, inhibition of PI 3-kinase by LY294002 or expression of a dominant interfering form of PI 3-kinase was sufficient to induce apoptosis even when cortical neurons were maintained under normal culture conditions in the presence of serum. These data suggest that the ERK pathway is primarily responsible for BDNF neuroprotection against the DNA-damaging agent camptothecin, whereas activation of the PI 3-kinase pathway contributes to BDNF neuroprotection against serum withdrawal and serum-promoted cortical neuron survival.
      These data demonstrate that multiple survival pathways are used in cortical neurons to counteract different forms of apoptotic signals, which probably occurs because different insults activate distinct biochemical pathways. For example, SCG neuron apoptosis induced by NGF withdrawal or DNA-damaging agents requires induction of different caspases as well as cyclin-dependent kinases (
      • Park D.S.
      • Morris E.J.
      • Padmanabhan J.
      • Shelanski M.L.
      • Geller H.M.
      • Greene L.A.
      ,
      • Park D.S.
      • Morris E.J.
      • Stefanis L.
      • Troy C.M.
      • Shelanski M.L.
      • Geller H.M.
      • Greene L.A.
      ,
      • Troy C.M.
      • Stefanis L.
      • Prochiantz A.
      • Greene L.A.
      • Shelanski M.L.
      ,
      • Troy C.M.
      • Stefanis L.
      • Greene L.A.
      • Shelanski M.L.
      ). Our data also emphasize the importance of defining apoptotic mechanisms for specific types of neuron, because neurons from different regions of the brain have distinct properties. Although activation of the ERK pathway does not contribute to the neuroprotective effect of NGF for SCG or BDNF in cultured cerebellar neurons (
      • Miller T.M.
      • Tansey M.G.
      • Johnson E.M.
      • Creedon D.J.
      ,
      • Virdee K.
      • Tolkovsky A.M.
      ,
      • Creedon D.J.
      • Johnson E.M.
      • Lawrence J.C.
      ,
      • Gunn Moore F.J.
      • Williams A.G.
      • Toms N.J.
      • Tavar'e J.M.
      ), our results demonstrate that it is neuroprotective for camptothecin-treated cortical neurons. Although activation of the PI 3-kinase/Akt pathway has been suggested to play a major role in neuroprotection by neurotrophic factors in cerebellar and SCG neurons (
      • Yao R.
      • Cooper G.M.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ,
      • D'Mello S.R.
      • Borodezt K.
      • Soltoff S.P.
      ,
      • Miller T.M.
      • Tansey M.G.
      • Johnson E.M.
      • Creedon D.J.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.J.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ), it has only a small effect on BDNF-mediated neuroprotection against camptothecin in cortical neurons.
      We also discovered that ERK was activated, rather than inhibited, when cortical neurons were treated with camptothecin and that inhibition of ERK activation by PD98059 further increased camptothecin-induced apoptosis. These findings suggest that ERK does not actively contribute to apoptosis but may be activated as a neuroprotective mechanism. Although the extent of ERK activation by camptothecin was small (2-fold), ERK activation lasted at least 24 h. This prolonged ERK activation may be critical for neuron survival because it provides a long window of opportunity for the activated ERK to transmit signals to downstream targets. Our data suggest the interesting possibility that ERK may function generally as a cell-intrinsic survival pathway in neurons and that this mechanism may apply to other forms of apoptosis in CNS neurons and in non-neuronal cells. For example, ERK is activated by H2O2 and low doses of radiation in several cell lines and may provide protection against these stimuli (
      • Carter S.
      • Auer K.L.
      • Reardon D.B.
      • Birrer M.
      • Fisher P.B.
      • Valerie K.
      • SchmidtUllrich R.
      • Mikkelsen R.
      • Dent P.
      ,
      • Guyton K.Z.
      • Liu Y.
      • Gorospe M.
      • Xu Q.
      • Holbrook N.J.
      ). Furthermore, ERK may be only one of several cellular self-defense mechanisms that are mobilized to combat stress. Another example is the activation of the NF-κB pathway in response to several forms of stress (
      • Beg A.A.
      • Baltimore D.
      ,
      • VanAntwerp D.J.
      • Martin S.J.
      • Kafri T.
      • Green D.R.
      • Verma I.M.
      ,
      • Wang C.Y.
      • Mayo M.W.
      • Baldwin A.S.
      ).
      The use of ERK as a cell survival mechanism in neurons is also interesting because ERK plays a pivotal role for other neuronal functions. For example, the ERK/MAP kinase signal transduction pathway is activated and required for transcriptionally dependent long-term potentiation as well as Ca2+ stimulation of the cAMP regulatory element transcriptional pathway in CNS neurons (
      • Impey S.
      • Obrietan K.
      • Wong S.T.
      • Poser S.
      • Yano S.
      • Wayman G.
      • Deloulme J.C.
      • Chan G.
      • Storm D.R.
      ). The activation of ERK and cAMP regulatory element-dependent transcription have been implicated in growth, differentiation, and neuroplasticity (
      • Mansour S.J.
      • Matten W.T.
      • Hermann A.S.
      • Candia J.M.
      • Rong S.
      • Fukasawa K.
      • Vande Woude G.F.
      • Ahn N.G.
      ,
      • Impey S.
      • Obrietan K.
      • Wong S.T.
      • Poser S.
      • Yano S.
      • Wayman G.
      • Deloulme J.C.
      • Chan G.
      • Storm D.R.
      ,
      • Qui M.S.
      • Green S.H.
      ). The dual function of ERK as a survival pathway and to mediate activity-dependent processes ensures the viability of neurons contributing to synaptic plasticity, and it may be an important mechanism for activity-dependent maintenance of neuron populations during development.
      In summary, the Erk and PI 3-kinase signaling pathways differentially mediate BDNF neuroprotection against camptothecin and serum deprivation. Our data suggest that signaling pathways that mediate neuroprotection are both stimulus- and cell-type specific.

      Acknowledgments

      We thank X. Masot, S. Impey, and W. Watt for critical reading of the manuscript. We also thank N. G. Ahn for providing MKK1 plasmids.

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