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

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 microM H2O2 elicited calcium signals, increased ryanodine receptor S-glutathionylation, and enhanced both ERK and CREB phosphorylation. In mouse hippocampal slices, H2O2 (1 microM) also stimulated ERK and CREB phosphorylation. Preincubation with ryanodine (50 microM) 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 microM 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.     

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

In vivo nicotine treatment regulates mesocorticolimbic CREB and ERK signaling in C57Bl/6J mice

The extracellular regulated kinase (ERK) pathway was studied to determine its role in neuronal plasticity related to the development of nicotine dependence. Levels and phosphorylation state of ERK, cAMP response element binding protein (CREB) and proline-rich/Ca2+-activated tyrosine kinase (PYK2), and levels of tyrosine hydroxylase (TH), were determined using western blotting. C57Bl/6J mice received acute or chronic nicotine (200 microg/mL) in their drinking water or were withdrawn from nicotine for 24 h following chronic exposure. CREB phosphorylation was reduced in the nucleus accumbens following chronic nicotine, consistent with previous reports that decreased accumbens CREB activity increases drug reinforcement. In contrast, CREB phosphorylation was increased in the prefrontal cortex following chronic nicotine exposure and in the ventral tegmental area during nicotine withdrawal. In addition, total and phosphorylated ERK decreased in the amygdala following chronic nicotine exposure, but ERK phosphorylation increased in the prefrontal cortex. TH levels increased in both the amygdala and prefrontal cortex, supporting the hypothesis that increased catecholaminergic tone contributes to nicotine reinforcement. Overall, these results support a role for ERK and CREB activity in neural plasticity associated with nicotine dependence.     

Serotonin mediates cross-modal reorganization of cortical circuits

Loss of one type of sensory input can cause improved functionality of other sensory systems. Whereas this form of plasticity, cross-modal plasticity, is well established, the molecular and cellular mechanisms underlying it are still unclear. Here, we show that visual deprivation (VD) increases extracellular serotonin in the juvenile rat barrel cortex. This increase in serotonin levels facilitates synaptic strengthening at layer 4 to layer 2/3 synapses within the barrel cortex. Upon VD, whisker experience leads to trafficking of the AMPA-type glutamate receptors (AMPARs) into these synapses through the activation of ERK and increased phosphorylation of AMPAR subunit GluR1 at the juvenile age when natural whisker experience no longer induces synaptic GluR1 delivery. VD thereby leads to sharpening of the functional whisker-barrel map at layer 2/3. Thus, sensory deprivation of one modality leads to serotonin release in remaining modalities, facilitates GluR1-dependent synaptic strengthening, and refines cortical organization.     

Elevated synaptic activity preconditions neurons against an in vitro model of ischemia

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.     

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.     

MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.     

Altered signaling pathways underlying abnormal hippocampal synaptic plasticity in the Ts65Dn mouse model of Down syndrome

The Ts65Dn mouse model of Down syndrome (DS) has an extra segment of chromosome (Chr.) 16 exhibits abnormal behavior, synaptic plasticity and altered function of several signaling molecules. We have further investigated signaling pathways that may be responsible for the impaired hippocampal plasticity in the Ts65Dn mouse. Here we report that calcium/calmodulin-dependent protein kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (ERK), protein kinase A (PKA) and protein kinase C (PKC), all of which have been shown to be involved in synaptic plasticity, are altered in the Ts65Dn hippocampus. We found that the phosphorylation of CaMKII and protein kinase Akt was increased, whereas ERK was decreased. Activities of PKA and PKC were decreased. Furthermore, abnormal PKC activity and an absence of the increase in Akt phosphorylation were demonstrated in the Ts65Dn hippocampus after high-frequency stimulation that induces long-term potentiation. Our findings suggest that abnormal synaptic plasticity in the Ts65Dn hippocampus is the result of compensatory alterations involving the glutamate receptor subunit GluR1 in either one or more of these signaling cascades caused by the expression of genes located on the extra segment of Chr. 16.     

Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival-promoting effects of BDNF and IGF-1 on serum deprivation-induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF-1 in the neurons. We found that the survival-promoting effect of BDNF was much weaker than that of IGF-1 in serum deprivation-induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3-kinase (PtdIns3-K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF-1 in the cultured cortical neurons, although many reports suggest that PtdIns3-K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen-activated protein kinase (MAPK) and cAMP responsive element-binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF-1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3-K-Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival-promoting effects of BDNF and IGF-1.     

Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic effects of flavanones in cortical neurons

There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone hesperetin and two of its metabolites, hesperetin-7- O -β- d -glucuronide and 5-nitro-hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst hesperetin glucuronide failed to exert protection, both hesperetin and 5-nitro-hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.     

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.