Elsevier

Cell Calcium

Volume 41, Issue 5, May 2007, Pages 491-502
Cell Calcium

Calcium release by ryanodine receptors mediates hydrogen peroxide-induced activation of ERK and CREB phosphorylation in N2a cells and hippocampal neurons

https://doi.org/10.1016/j.ceca.2006.10.001Get rights and content

Abstract

Hydrogen peroxide, which stimulates ERK phosphorylation and synaptic plasticity in hippocampal neurons, has also been shown to stimulate calcium release in muscle cells by promoting ryanodine receptor redox modification (S-glutathionylation). We report here that exposure of N2a cells or rat hippocampal neurons in culture to 200 μM H2O2 elicited calcium signals, increased ryanodine receptor S-glutathionylation, and enhanced both ERK and CREB phosphorylation. In mouse hippocampal slices, H2O2 (1 μM) also stimulated ERK and CREB phosphorylation. Preincubation with ryanodine (50 μM) largely prevented the effects of H2O2 on calcium signals and ERK/CREB phosphorylation. In N2a cells, the ERK kinase inhibitor U0126 suppressed ERK phosphorylation and abolished the stimulation of CREB phosphorylation produced by H2O2, suggesting that H2O2 enhanced CREB phosphorylation via ERK activation. In N2a cells in calcium-free media, 200 μM H2O2 stimulated ERK and CREB phosphorylation, while preincubation with thapsigargin prevented these enhancements. These combined results strongly suggest that H2O2 promotes ryanodine receptors redox modification; the resulting calcium release signals, by enhancing ERK activity, would increase CREB phosphorylation. We propose that ryanodine receptor stimulation by activity-generated redox species produces calcium release signals that may contribute significantly to hippocampal synaptic plasticity, including plasticity that requires long-lasting ERK-dependent CREB phosphorylation.

Introduction

Activity-dependent phosphorylation of the transcription factor cAMP/Ca2+ response element binding protein (CREB) induces the transcription of several neuronal genes [43], [62]. CREB phosphorylation is considered critical to induce long-term potentiation (LTP) and for several forms of learning and memory [10], [41], [56]. CREB-dependent transcription of genes involved in synaptic plasticity entails long-term CREB phosphorylation by the Ca2+-sensitive Ras/ERK (extracellular signal-regulated kinase) pathway [25], [63]. Most studies on Ca2+-dependent neuronal gene expression have focused on neuronal Ca2+ entry pathways. Yet, Ca2+ release from intracellular stores also contributes to activity-dependent gene expression [37], [50], [57]. In particular, Ca2+ release by ryanodine receptors (RyR) contributes to synaptic plasticity and neuronal gene expression [4], [6], [22], [40], [55].

Functional RyR are required for long-lasting long-term potentiation (LTP) and for activity-dependent increases in phosphorylated CREB (phospho-CREB) in hippocampal area CA1 postsynaptic neurons [40]. RyR activity is highly sensitive to direct redox modification by reactive oxygen and nitrogen species (ROS/RNS) [2], [20], [21], [28], [46]. Active neurons display increased metabolic activity and oxygen consumption, as well as increased generation of ROS/RNS [15], [64]; moreover, ROS generation has been implicated on hippocampal LTP [54]. Cell-permeable scavengers of superoxide anion, a free radical, block LTP induction in hippocampal area CA1 [36], a region which also contains a ROS producing NADPH oxidase (NOX) that is required for N-methyl-d-aspartate (NMDA) receptor-dependent ERK activation [35], [53], [58]. NOX-generated superoxide anion dismutates into H2O2, a ROS that at low concentrations (1 μM) increases tetanic LTP 2-fold and also enhances NMDA-independent LTP [32], [33]. Interestingly, catalase, which scavenges H2O2, attenuates LTP [59]. Although electrophysiological studies have yielded divergent results on the effects of H2O2 on hippocampal function, in some studies the use of non-physiological H2O2 concentrations in the mM range may have caused deleterious oxidative reactions unrelated to the potential physiological responses [33].

We investigated here whether RyR channels participate in H2O2-induced ERK phosphorylation in N2a cells or hippocampal neurons. We found that H2O2 modified RyR redox state, increasing its S-glutathionylation. H2O2 also stimulated Ca2+ release and increased sequentially ERK and CREB phosphorylation, while specific RyR inhibition by 50 μM ryanodine drastically reduced the stimulation of Ca2+ release and of ERK/CREB phosphorylation induced by H2O2. We propose that ROS generated during hippocampal LTP induction stimulate RyR, enhancing Ca2+ release and the Ca2+-dependent ERK/CREB phosphorylation cascade required for CREB-dependent gene transcription.

Section snippets

Cell cultures

Cell culture media were obtained from InVitrogen (Grand Island, NY). Mouse neuroblastoma (N2a) cells (CCL-131, American Type Culture Collection, Rockville, MD), were plated on 35 mm culture dishes in Dulbecco's modified Eagle medium supplemented with 2 mM l-glutamine, 110 mg/l sodium pyruvate and pyridoxine hydrochloride adjusted to contain 3.7 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 5% fetal bovine serum, antibiotics and antimycotics, and maintained at 37 °C. The culture medium was

Results

Activation of the Ras/ERK pathway is required for long-term CREB phosphorylation (Wu et al., 2001) [60], a prerequisite of sustained, long-lasting LTP in the hippocampus [41], [56]. Accordingly, the study of the cellular factors that may affect CREB phosphorylation via the ERK pathway in hippocampal neurons is of special significance. In this work, we investigated how modifying the cellular redox state with H2O2 affected ERK and CREB phosphorylation in neuronal cells in culture or in

Discussion

The results described herein suggest strongly that stimulation of RyR-mediated Ca2+ release, presumably through H2O2-induced redox modifications of the RyR protein, is responsible for the ERK/CREB activation displayed by N2a cells and hippocampal neurons exposed to H2O2. The physiological relevance of these results lies in their possible relevance to long-lasting LTP, which in the hippocampus requires ROS production and the Ca2+-induced Ras/ERK activation necessary for long-lasting CREB

Acknowledgements

The contribution of C. Sofía Hernández in exploratory experiments and the technical help provided by Mónica Silva, Nancy Leal, Luis Montecinos and Laura Villasana are gratefully acknowledged. This study was supported by FONDAP Center for Molecular Studies of the Cell, Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT) grant 15010006, by FONDECYT grant 1030988, and by the National Institutes of Health (NS34007).

References (67)

  • G.E. Hardingham et al.

    The yin and yang of NMDA receptor signalling

    Trends Neurosci.

    (2003)
  • J. Hongpaisan et al.

    Calcium-dependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons

    Mol. Cell Neurosci.

    (2003)
  • T. Imagawa et al.

    Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

    J. Biol. Chem.

    (1987)
  • B.E. Lonze et al.

    Function and regulation of CREB family transcription factors in the nervous system

    Neuron

    (2002)
  • J.J. Marengo et al.

    Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells

    Biophys. J.

    (1998)
  • M.T. Nuñez et al.

    Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

    Free Radic. Biol. Med.

    (2004)
  • P. Puttfarcken et al.

    Inhibition of age-induced beta-amyloid neurotoxicity in rat hippocampal cells

    Exp. Neurol.

    (1996)
  • C.R. Rose et al.

    Stores not just for storage: intracellular calcium release and synaptic plasticity

    Neuron

    (2001)
  • G. Sánchez et al.

    Tachycardia increases NADPH oxidase activity and RyR2 S-glutathionylation in ventricular muscle

    J. Mol. Cell. Cardiol.

    (2005)
  • F.Q. Schafer et al.

    Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple

    Free Radic. Biol. Med.

    (2001)
  • F. Serrano et al.

    NADPH oxidase immunoreactivity in the mouse brain

    Brain Res.

    (2003)
  • F. Serrano et al.

    Reactive oxygen species and synaptic plasticity in the aging hippocampus

    Ageing Res. Rev.

    (2004)
  • M. Shimuta et al.

    Postsynaptic modulation of AMPA receptor-mediated synaptic responses and LTP by the type 3 ryanodine receptor

    Mol. Cell. Neurosci.

    (2001)
  • K. Svoboda et al.

    [Ca2+]: intracelular stores spill their guts

    Neuron

    (1999)
  • M.V. Tejada-Simon et al.

    Synaptic localization of a functional NADPH oxidase in the mouse hippocampus

    Mol. Cell Neurosci.

    (2005)
  • L. Zhang et al.

    Oxidative stress differentially modulates phosphorylation of ERK, p38 and CREB induced by NGF or EGF in PC12 cells

    Neurobiol. Aging

    (1999)
  • A. Balkowiec et al.

    Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons

    J. Neurosci.

    (2002)
  • D. Balschun et al.

    Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning

    EMBO J.

    (1999)
  • B. Bedogni et al.

    Redox regulation of CREB and induction of manganese superoxide dismutase in NGF-dependent cell survival

    J. Biol. Chem.

    (2003)
  • V.P. Bindokas et al.

    Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine

    J. Neurosci.

    (1996)
  • R. Bull et al.

    SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP

    Am. J. Physiol. Cell Physiol.

    (2003)
  • M.A. Carrasco et al.

    Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells

    Biol. Res.

    (2004)
  • D.M. Chetkovich et al.

    Nitric oxide synthase-independent long-term potentiation in area CA1 of hippocampus

    Neuroreport

    (1993)
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