Extracellular Signal-regulated Kinase In Neurons Research Articles

Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

Intracranial self-stimulation-reward or immobilization-aversion had different effects on neurite extension and the ERK pathway in neurotransmitter-sensitive mutant PC12 cells

Various actions trigger pleasure (reward) or aversion (punishment) as emotional responses. Emotional factors that negatively affect brain neural control processes for long periods of time might cause various mental diseases by inducing neuronal changes. In the present study, newly developed PC12m12 cells which are highly sensitivity to neurotransmitters such as acetylcholine (ACh), were used. Exposing the cells to plasma from rats that had been subjected to intracranial self-stimulation (ICSS) markedly upregulated neurite outgrowth. In addition, voluntary running in a wheel or forced on a rotating rod was used to induce behavioral excitation in rats, and examinations of their plasma confirmed that the ICSS-induced neurite outgrowth was not associated with the ICSS behavior itself. Furthermore, immunoblotting and treatment with U0126, an ERK (extracellular signal-regulated kinase) antagonist, showed that the ICSS-induced neurite outgrowth was related to neuronal ERK activity. Exposing the same cells to plasma from rats that had been subjected to immobilization (IMM) also increased neurite outgrowth. Although the degree of enhancement was not as great as that seen after the ICSS rat plasma treatment, it was less than that observed after treatment with ACh as a positive control. These results indicate that ICSS or IMM lead to varying degrees of morphological changes, such as enhanced neurite outgrowth, in PC12m12 cells, but the neuronal signal transduction pathways underlying these effects differ; i.e.,the former morphological change might involve the activation of the ERK pathway, whereas the latter changes might not. Using PC12m12 cells which exhibit sensitivity to neurotransmitters, it might be possible to clarify the pathogeneses of mental diseases at the neuronal level and search for therapeutic drugs.

Is Histidine promising in alleviating the risk of Metabolic disorder?

AbstractIntroductionIn recent times, evidence from published reports; randomized clinical trial (RCT), human case-controls, metabolomics, animal and cell studies is gaining rapt significance on the ameliorative potential of histidine (His) in obesity/insulin resistance (IR)/ inflammation outcomes. This study aimed at systematically mining evidence from previously published studies on the vitality of His on metabolic disorder outcomes.MethodsUsing the MOOSE guidelines we searched PubMed and Cochrane library using the search words “histidine” and “obesity” or “diabetes” or “metabolic syndrome” and included articles on His intake/administration and obesity/diabetes/metabolic disorders involving human and/or animal experimentation.ResultsWe found low circulating His profile has been associated with metabolic disorders independent of ethnicity [1–4]. Similarly, two separate animal trials [5–6] found His to be significantly lower in diabetic Balb/cA mice and lean Zucker rat. Also, the endogenous absence of His disrupts mitochondrial membrane integrity thereby impairing energy production [7]. In addition, epidemiological report revealed higher dietary His was associated with lower overweight and obesity risk [8]. Though epidemiological reports from the diverse population to assert this observation are insufficient, reports from animal models [9–10] revealed similar conclusions. Also, some reports [11–12] have demonstrated the dose-dependent inhibitory effect of His on some inflammatory markers by assuaging the palmitic acid induced pAkt/Akt expression [12]. Also, His inhibits the up-production of free radicals to impede the extracellular signal-regulated kinase in neurons [13].ConclusionThese evidences are novel but primarily limited to mostly obesity. In our opinion, future multi-ethnic longitudinal cohort and trials should consider exploring effects of His on non-fatty alcoholic liver diseases and cardiovascular complications. His might as well be beneficial potentially offering significant hope and non-invasive alternatives in the management of metabolic disorders.Note:Complete citations available inMEDLINE;[1] PMID: 20834187 [2] PMID: 22266733 [3] PMID: 21996294 [4] PMID: 23697717 [5] PMID:15878720 [6] PMID:15389298 [7] PMID: 4621606 [8] PMID: 27409634 [9] PMID: 17372311 [10] PMID: 15561489 [11] PMID: 16107255 [12] PMID: 23361591 [13] PMID: 22101981

Sustained stimulation of β2- and β3-adrenergic receptors leads to persistent functional pain and neuroinflammation

Functional pain syndromes, such as fibromyalgia and temporomandibular disorder, are associated with enhanced catecholamine tone and decreased levels of catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines). Consistent with clinical syndromes, our lab has shown that sustained 14-day delivery of the COMT inhibitor OR486 in rodents results in pain at multiple body sites and pain-related volitional behaviors. The onset of COMT-dependent functional pain is mediated by peripheral β2- and β3-adrenergic receptors (β2- and β3ARs) through the release of the pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Here, we first sought to investigate the role of β2- and β3ARs and downstream mediators in the maintenance of persistent functional pain. We then aimed to characterize the resulting persistent inflammation in neural tissues (neuroinflammation), characterized by activated glial cells and phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK). Separate groups of rats were implanted with subcutaneous osmotic mini-pumps to deliver OR486 (15 mg/kg/day) or vehicle for 14 days. The β2AR antagonist ICI118551 and β3AR antagonist SR59230A were co-administrated subcutaneously with OR486 or vehicle either on day 0 or day 7. The TNFα inhibitor Etanercept, the p38 inhibitor SB203580, or the ERK inhibitor U0126 were delivered intrathecally following OR486 cessation on day 14. Behavioral responses, pro-inflammatory cytokine levels, glial cell activation, and MAPK phosphorylation were measured over the course of 35 days. Our results demonstrate that systemic delivery of OR486 leads to mechanical hypersensitivity that persists for at least 3 weeks after OR486 cessation. Corresponding increases in spinal TNFα, IL-1β, and IL-6 levels, microglia and astrocyte activation, and neuronal p38 and ERK phosphorylation were observed on days 14–35. Persistent functional pain was alleviated by systemic delivery of ICI118551 and SR59230A beginning on day 0, but not day 7, and by spinal delivery of Etanercept or SB203580 beginning on day 14. These results suggest that peripheral β2- and β3ARs drive persistent COMT-dependent functional pain via increased activation of immune cells and production of pro-inflammatory cytokines, which promote neuroinflammation and nociceptor activation. Thus, therapies that resolve neuroinflammation may prove useful in the management of functional pain syndromes.

Inhibition of basal and amphetamine-stimulated extracellular signal-regulated kinase (ERK) phosphorylation in the rat forebrain by muscarinic acetylcholine M4 receptors

The mitogen-activated protein kinase (MAPK), especially its extracellular signal-regulated kinase (ERK) subfamily, is a group of kinases enriched in the mammalian brain. While ERK is central to cell signaling and neural activities, the regulation of ERK by transmitters is poorly understood. In this study, the role of acetylcholine in the regulation of ERK was investigated in adult rat striatum in vivo. We focused on muscarinic M1 and M4 receptors, two principal muscarinic acetylcholine (mACh) receptor subtypes in the striatum. A systemic injection of the M1-preferring antagonist telenzepine did not alter ERK phosphorylation in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. Similarly, telenzepine did not affect ERK phosphorylation in the medial prefrontal cortex (mPFC), hippocampus, and cerebellum. Moreover, telenzepine had no effect on the ERK phosphorylation induced by dopamine stimulation with the psychostimulant amphetamine. In contrast to telenzepine, the M4-preferring antagonist tropicamide consistently increased ERK phosphorylation in the striatum and mPFC. This increase was rapid and transient. Tropicamide and amphetamine when coadministered at subthreshold doses induced a significant increase in ERK phosphorylation. These results demonstrate that mACh receptors exert a subtype-specific modulation of ERK in striatal and mPFC neurons. While the M1 receptor antagonist has no effect on ERK phosphorylation, M4 receptors inhibit constitutive and dopamine-stimulated ERK phosphorylation in these dopamine-innervated brain regions.

Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling can increase guinea pig cardiac neuron excitability in part through extracellular signal-regulated kinase (ERK) activation. The present study examined the PACAP receptors and signaling cascades that stimulate guinea pig cardiac neuron ERK signaling using confocal microscopy to quantify PACAP-induced neuronal phosphorylated ERK (pERK) immunoreactivity. PACAP and maxadilan, but not vasoactive intestinal polypeptide (VIP), increased cardiac neuron pERK, implicating primary roles for PACAP-selective PAC1 receptor (Adcyap1r1) signaling rather than VPAC receptors (Vipr1 and Vipr2) in the generation of cardiac neuron pERK. The adenylyl cyclase (AC) activator forskolin, but not the protein kinase C (PKC) activator phorbol myristate acetate (PMA), increased pERK. Also, Bim1 did not blunt PACAP activation of pERK. Together, the results suggest PAC1 receptor signal transduction via Gs/adenylyl cyclase (AC)/cAMP rather than Gq/phospholipase C (PLC) generated neuronal pERK. Activator and inhibitor studies suggested that the PACAP-mediated pERK activation was PKA-dependent rather than an exchange protein directly activated by a cAMP (EPAC), PKA-independent mechanism. The PACAP-induced pERK was inhibited by the clathrin inhibitor Pitstop2 to block receptor internalization and endosomal signaling. We propose that the PACAP-mediated MEK/ERK activation in cardiac neurons involves both AC/cAMP/PKA signaling and PAC1 receptor internalization/activation of signaling endosomes.

Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction.

Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p < 0.0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a "switchlike" fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11.3% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2-13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local collagen fiber kinematics, may be associated with mechanotransduction signaling in neurons.

Ginsenoside Rb1 Attenuates Acute Inflammatory Nociception by Inhibition of Neuronal ERK Phosphorylation by Regulation of the Nrf2 and NF-κB Pathways.

Ginsenoside-Rb1 (Rb1) has anti-inflammatory effects. However, the potential antinociceptive value of Rb1 for the treatment of acute inflammatory nociception is still unknown. In this study, we examined whether Rb1 has any antinociceptive effects on acute inflammatory nociception in Sprague Dawley rats given intrathecal (i.t.) introduction of Rb1 (2, 10, and 50μg) 20minutes before injection of formalin (5%, 50μL) into the plantar surface of the hind paws. I.t. introduction of Rb1 significantly decreased nociceptive behavior during phase II (16-60minutes), but not phase I (0-10minutes), after formalin stimulation, corresponding to the reduced activation of c-Fos in the L4 to L5 spinal dorsal horn after formalin stimulation. Rb1 also reduced the phosphorylation of extracellular signal-regulated kinase in the neurons, but not the microglia and astrocytes. Microscopic examination of the microglia and astrocytes revealed no morphological changes due to formalin stimulation and i.t. introduction of Rb1. Interestingly, Rb1 activated the nuclear factor erythroid 2-related factor 2 pathway and inhibited nuclear factor kappa B pathways. Our findings indicate that i.t. introduction of Rb1 might effectively inhibit formalin-induced acute inflammatory nociception by inhibition of neuronal extracellular signal-regulated kinase phosphorylation, which is thought to regulate the nuclear factor erythroid 2-related factor 2 nuclear factor kappa B pathways in the spinal dorsal horn, which suggests therapeutic potential for suppression of acute inflammatory pain.

Neuronal extracellular signal-regulated kinase (ERK) activity as marker and mediator of alcohol and opioid dependence

Early pioneering work in the field of biochemistry identified phosphorylation as a crucial post-translational modification of proteins with the ability to both indicate and arbitrate complex physiological processes. More recent investigations have functionally linked phosphorylation of extracellular signal-regulated kinase (ERK) to a variety of neurophysiological mechanisms ranging from acute neurotransmitter action to long-term gene expression. ERK phosphorylation serves as an intracellular bridging mechanism that facilitates neuronal communication and plasticity. Drugs of abuse, including alcohol and opioids, act as artificial yet powerful rewards that impinge upon natural reinforcement processes critical for survival. The graded progression from initial exposure to addiction (or substance dependence) is believed to result from drug- and drug context-induced adaptations in neuronal signaling processes across brain reward and stress circuits following excessive drug use. In this regard, commonly abused drugs as well as drug-associated experiences are capable of modifying the phosphorylation of ERK within central reinforcement systems. In addition, chronic drug and alcohol exposure may drive ERK-regulated epigenetic and structural alterations that underlie a long-term propensity for escalating drug use. Under the influence of such a neurobiological vulnerability, encountering drug-associated cues and contexts can produce subsequent alterations in ERK signaling that drive relapse to drug and alcohol seeking. Current studies are determining precisely which molecular and regional ERK phosphorylation-associated events contribute to the addiction process, as well as which neuroadaptations need to be targeted in order to return dependent individuals to a healthy state.

Relationship between extracellular signal-regulated kinase and ketamine-induced apoptosis in rat hippocampal neurons

Objective To evaluate the relationship between extracellular signal-regulated kinase (ERK)and ketamine-induced apoptosis in rat hippocampal neurons.Methods Sprague-Dawley rats at 18 days of gestation were anesthetized.The fetal rats were obtained under the sterile condition and decapitated.The hippocampal neurons were isolated and primarily cultured for 5 days,and were seeded in 6-well plates (2 ml/well) or in 96-well plates (100μl/well) at a density of 5 × 105/ml.The cells were randomly divided into 4 groups (n =18 each):control group (group C),fibroblast growth factor (FGF-2,an ERK agonist) group (group F),ketamine group (group K) and FGF-2 + ketamine group (group FK).The cells were cultured in the plain culture medium in group C.FGF-2 50 ng/ml was added to the culture medium in group F.Ketamine was added to the culture medium in group K.FGF-2 50 ng/ml was added to the culture medium at 20 min before ketamine 100 μmol/L was added in group FK.The phosphorylation of ERK in hippocampal neurons was detected by Western blot at 10 min after treatment.At 24 h after treatment,the neuronal apoptosis was detected by Hoechst33342/PI staining,and the cell survival rate was detected by MTT assay.The apoptosis rate was calculated.Results Compared with group C,the phosphorylation of ERK in hippocampal neurons and the cell survival rate was significantly decreased and the apoptosis rate was increased in K and FK groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between F and C groups (P ＞ 0.05).The phosphorylation of ERK in hippocampal neurons and the cell survival rat was significantly higher and the apoptosis rate was lower in group FK than in group K (P ＜0.05).Conclusion Ketamine induces apoptosis in rat hippocampal neurons by inhibiting activation of ERK in hippocampal neurons. Key words: Extracellular signal-regulated MAP kinases; Ketamine; Hippocampus ; Neuron ; Apoptosis

Analgesic effects of the COX-2 inhibitor parecoxib on surgical pain through suppression of spinal ERK signaling

Cyclooxygenase (COX)-2 inhibitors are widely used for postoperative pain control in clinical practice. However, it is unknown whether spinal sensitization is involved in the analgesic effects of COX-2 inhibitors on surgical pain. Extracellular signal-regulated kinase (ERK) in the spinal cord is implicated in various types of pain, including surgical pain. The present study investigated the role of spinal ERK signaling in the analgesic effect of the COX-2 inhibitor parecoxib on surgical pain. Surgical pain was produced in rats by surgical incision of the hind paw. Phosphorylated (p)-ERK1/2 expression was determined by immunohistochemistry. Pain hypersensitivity was evaluated by measuring the paw withdrawal threshold using the von Frey test. The selective COX-2 inhibitor parecoxib was delivered 20 min before or 20 min after the incision by intraperitoneal injection. Pretreatment with parecoxib markedly attenuated the pain hypersensitivity induced by incision. However, post-treatment with parecoxib produced minimal analgesic effects. Parecoxib inhibited the increase in spinal p-ERK expression following surgical incision. The present study thus suggests that the COX-2 inhibitor parecoxib exerts its analgesic effect on surgical pain through the inhibition of neuronal ERK activation in the spinal cord. COX-2 inhibitor delivery prior to surgery has more potent analgesic effects, suggesting the advantage of preventive analgesia for post-operative pain control.

Cortical effect of oxaliplatin associated with sustained neuropathic pain: Exacerbation of cortical activity and down-regulation of potassium channel expression in somatosensory cortex

Oxaliplatin is a third-generation platinum-based chemotherapy drug that has gained importance in the treatment of advanced metastatic colorectal cancer. Its dose-limiting side effect is the production of chronic peripheral neuropathy. Using a modified model of oxaliplatin-induced sensory neuropathy, we investigated plastic changes at the cortical level as possible mechanisms underlying the chronicity of pain sensation in this model. Changes in gene expression were studied using DNA microarray which revealed that when oxaliplatin-treated animals displayed clinical neuropathic pain symptoms, including mechanical and thermal hypersensitivity, approximately 900 were down-regulated in the somatosensory cortex. Because of the known role of potassium channels in neuronal excitability, the study further focussed on the down-regulation of these channels as the possible molecular origin of cortical hyperexcitability. Quantification of the magnitude of neuronal extracellular signal-regulated kinase (ERK) phosphorylation in cortical neurons as a marker of neuronal activity revealed a 10-fold increase induced by oxaliplatin treatment, suggesting that neurons of cortical areas involved in transmission of painful stimuli undergo a chronic cortical excitability. We further demonstrated, using cortical injection of lentiviral vector shRNA against Kv2.2, that down-regulation of this potassium channel in naive animals induced a sustained thermal and mechanical hypersensitivity. In conclusion, although the detailed mechanisms leading to this cortical excitability are still unknown, our study demonstrated that a cortical down regulation of potassium channels could underlie pain chronicity in this model of chemotherapy-induced neuropathic pain.

Ethyl Pyruvate Attenuates Formalin-Induced Inflammatory Nociception by Inhibiting Neuronal ERK Phosphorylation

BackgroundEthyl pyruvate (EP) possesses anti-inflammatory activity. However, the potential anti-nociceptive value of EP for the treatment of the inflammatory nociception is largely unknown. We investigated whether EP could have any anti-nociceptive effect on inflammatory pain, after systemic administration of EP (10, 50, and 100 mg/kg, i.p.), 1 hour before formalin (5%, 50 μl) injection into the plantar surface of the hind paws of rats.ResultsEP significantly decreased formalin-induced nociceptive behavior during phase II, the magnitude of paw edema, and the activation of c-Fos in L4-L5 spinal dorsal horn. EP also attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) in the neurons of L4-L5 spinal dorsal horn after formalin injection. Interestingly, the i.t. administration of PD98059, an ERK upstream kinase (MEK) inhibitor, completely blocked the formalin-induced inflammatory nociceptive responses.ConclusionsThese results demonstrate that EP may effectively inhibit formalin-induced inflammatory nociception via the inhibition of neuronal ERK phosphorylation in the spinal dorsal horn, indicating its therapeutic potential in suppressing acute inflammatory pain.

Sustained activation of ERK signaling in astrocytes is critical for neuronal injury‐induced monocyte chemoattractant protein‐1 production in rat corticostriatal slice cultures

We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.

NMDA-mediated and self-induced bdnf exon IV transcriptions are differentially regulated in cultured cortical neurons

Activity-dependent transcriptional up-regulation of bdnf (brain-derived neurotrophic factor) is involved in regulating many aspects of neuronal functions. The NMDA ( N-methyl- d-aspartic acid)-mediated and BDNF-mediated exon IV transcription may represent mechanistically different responses, and relevant to activity-dependent changes in neurons. We found that the activities of ERK (extracellular signal-regulated kinase), CaM KII/IV (calmodulin-dependent protein kinase II and IV), PI3K (phosphoinositide 3-kinase), and PLC (phospholipase C) are required for NMDA receptor-mediated bdnf exon IV transcription in cultured cortical neurons. In contrast, the BDNF-induced and TrkB-dependent exon IV transcription was regulated by ERK and CaM KII/IV, but not by PI3K and PLC. While ERK and CaM KII/IV are separate signaling pathways in BDNF-stimulated neurons, CaM KII/IV appeared to regulate exon IV transcription through ERK in NMDA-stimulated neurons. Similarly, the PI3K and PLC signaling pathways converged on ERK in NMDA- but not BDNF-stimulated neurons. Our results implicate that the NMDA-induced and the self-maintenance of bdnf transcription are differentially regulated.

MAP'ing CNS Development and Cognition: An ERKsome Process

The ERK MAP kinase signaling cascade plays critical roles in brain development, learning, memory, and cognition. It has recently been appreciated that mutation or deletion of elements within this signaling pathway leads to developmental syndromes in humans that are associated with impaired cognitive function and autism. Here, we review recent studies that provide insight into the biological roles of the ERKs in the brain that may underlie the cognitive deficits seen in these syndromes.

Activation of the Neuronal Extracellular Signal-Regulated Kinase 2 in the Spinal Cord Dorsal Horn Is Required for Complete Freund's Adjuvant-Induced Pain Hypersensitivity

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling in the spinal cord dorsal horn (SCDH) has been implicated in injury-induced pain hypersensitivity. Available ERK pathway inhibitors cannot distinguish between ERK1 and ERK2, nor can they differentially target the expression of neuronal or glial ERK1/2. We selectively inhibited the expression of ERK2 in neurons of the adult mouse SCDH by use of an ERK2 small interfering RNA (siRNA) delivered by a neurotropic adenoassociated viral vector. In situ hybridization revealed a siRNA vector-induced decrease in ERK2 mRNA in the ipsilateral SCDH. Immunohistochemistry showed a decreased neuronal phospho-ERK1/2 (pERK1/2), and Western blot analysis revealed that both ERK2 expression and phosphorylation were reduced by the siRNA vector. In contrast, basal ERK1 expression was not affected, although pERK1 was slightly increased. The siRNA vector-induced knockdown of ERK2 expression in the SCDH did not alter the baseline mechanical or thermal paw withdrawal thresholds. Hindpaw intraplantar injection of complete Freund's adjuvant (CFA) produced peripheral inflammation, mechanical allodynia, and thermal hyperalgesia in vector control animals that persisted for at least 96 h. It also caused an increase in SCDH ERK1 and ERK2 levels at 96 h and pERK1 and pERK2 levels at 1 and 96 h. The ERK2 siRNA vector prevented changes in ERK1, ERK2, and pERK2. In addition, the siRNA vector protected the animals from developing mechanical allodynia and thermal hyperalgesia throughout the 96 h after CFA. These findings indicate that ERK2 in the SCDH neurons is critical for the development of inflammatory pain hypersensitivity.

Protease‐activated receptor dependent and independent signaling by kallikreins 1 and 6 in CNS neuron and astroglial cell lines

While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.

Evidence of Aβ‐ and transgene‐dependent defects in ERK‐CREB signaling in Alzheimer’s models

Extracellular-signal regulated kinase (ERK) signaling is critical for memory and tightly regulated by acute environmental stimuli. In Alzheimer disease transgenic models, active ERK is shown to first be increased, then later reduced, but whether these baseline changes reflect disruptions in ERK signaling is less clear. We investigated the influence of the familial Alzheimer's disease transgene APPsw and beta-amyloid peptide (Abeta) immunoneutralization on cannulation injury-associated (i.c.v. infusion) ERK activation. At both 12 and 22 months of age, the trauma-associated activation of ERK observed in Tg(-) mice was dramatically attenuated in Tg(+). In cortices of 22-month-old non-infused mice, a reduction in ERK activation was observed in Tg(+), relative to Tg(-) mice. Intracerebroventricular (i.c.v.) anti-Abeta infusion significantly increased phosphorylated ERK, its substrate cAMP-response element-binding protein (CREB) and a downstream target, the NMDA receptor subunit. We also demonstrated that Abeta oligomer decreased active ERK and subsequently active CREB in human neuroblastoma cells, which could be prevented by oligomer immunoneutralization. Abeta oligomers also inhibited active ERK and CREB in primary neurons, in addition to reducing the downstream post-synaptic protein NMDA receptor subunit. These effects were reversed by anti-oligomer. Our data strongly support the existence of an APPsw transgene-dependent and Abeta oligomer-mediated defect in regulation of ERK activation.

Visual Stimulation Activates ERK in Synaptic and Somatic Compartments of Rat Cortical Neurons with Parallel Kinetics

BackgroundExtracellular signal-regulated kinase (ERK) signalling pathway plays a crucial role in regulating diverse neuronal processes, such as cell proliferation and differentiation, and long-term synaptic plasticity. However, a detailed understanding of the action of ERK in neurons is made difficult by the lack of knowledge about its subcellular localization in response to physiological stimuli. To address this issue, we have studied the effect of visual stimulation in vivo of dark-reared rats on the spatial-temporal dynamics of ERK activation in pyramidal neurons of the visual cortex.Methodology/Principal FindingsUsing immunogold electron microscopy, we show that phosphorylated ERK (pERK) is present in dendritic spines, both at synaptic and non-synaptic plasma membrane domains. Moreover, pERK is also detected in presynaptic axonal boutons forming connections with dendritic spines. Visual stimulation after dark rearing during the critical period causes a rapid increase in the number of pERK-labelled synapses in cortical layers I–II/III. This visually-induced activation of ERK at synaptic sites occurs in pre- and post-synaptic compartments and its temporal profile is identical to that of ERK activation in neuronal cell bodies.Conclusions/SignificanceVisual stimulation in vivo increases pERK expression at pre- and post-synaptic sites of axo-spinous junctions, suggesting that ERK plays an important role in the local modulation of synaptic function. The data presented here support a model in which pERK can have early and late actions both centrally in the cell nucleus and peripherally at synaptic contacts.