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