Neurotrophin-3 stimulates neurogenetic proliferation via the extracellular signal-regulated kinase pathway

The effects of neurotrophin-3 (NT3) administered into the ventricular space of 13.5-day-old mouse embryos on neurogenesis in the developing cerebral cortex were examined. 5-Bromo-2'-deoxyuridine (BrdU) was injected into pregnant mice 3 hr after the NT3 administration to label the neural progenitor cells. NT3 increased the number of BrdU-positive cells without altering their distribution. The increment in BrdU-positive cells 24 hr after the BrdU injection was attributed to Pax6-/BrdU-positive cells (neural stem cells), which was followed by a significant elevation of the number of Tuj1-/BrdU-positive cells (neurons) 36 or 48 hr after the BrdU injection, suggesting that NT3 facilitated neurogenesis by acting in two sequential steps, i.e., causing proliferation of neural stem cells and generation of neurons from these progenitors. NT3 stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 in the cortical progenitors, and the effects of NT3 on the increase in total BrdU-positive cells and Pax6-/BrdU-positive cells were diminished by an MEK inhibitor, suggesting the involvement of MEK-mediated ERK signal transduction in the NT3 actions.     

Estrogen stimulates the mitogen-activated protein kinase pathway in midbrain astroglia

Estrogen stimulates the development of midbrain dopamine neurons predominantly by acting through membrane receptors coupled to Ca(2+)-signaling. In this report, we describe that estrogen activates extracellular signal-regulated kinases (ERK1/2) in midbrain astrocytes but not neurons. This effect was inhibited by BAPTA which interrupts Ca(2+)-signaling but not by antagonists specific for other signaling pathways. The activation of the MAP kinase pathway suggests a potential role for astrocytes in mediating estrogen effects in the midbrain.     

The role of the extracellular signal-regulated kinase pathway in memory encoding

All types of memory depend on the integrated activity of various brain structures and neurotransmitter systems and involve more than one receptor, signal transduction pathway and postsynaptic mechanism. The components of the extracellular signal regulated kinases-1 and -2 (ERK1/2) signal transduction pathways are ubiquitous and well conserved protein kinases involved in relaying extracellular signals into intracellular responses, and are involved in the mechanisms of synaptic plasticity, learning and memory. ERK activation is required for the full expression of long-term potentiation (LTP), the principal cellular mechanism thought to underlie neuronal plasticity. Furthermore, ERK is activated in and is necessary for the development of several forms of memory, such as fear conditioning, conditioned taste aversion memory, spatial memory, step-down inhibitory avoidance and object recognition memory. ERK activation is secondary to neurotransmitter release and activation of the forebrain cholinergic neurons during and immediately after acquisition of an inhibitory avoidance response, revealed by increased release of acetylcholine (ACh), which in turn activates ERK in neurons located in the medial prefrontal cortex and ventral hippocampus. Increased release of ACh and ERK activation are events mechanistically related to each other, as demonstrated by the use of scopolamine, a muscarinic receptor antagonist, and by inhibitors of ERK activation, which blocked memory encoding and ERK activation. A critical function of activated ERK downstream of the increased ACh release occurring during learning is to promote cellular integration of divergent downstream effectors which may trigger different responses, depending upon which subsets of scaffolding anchors, target proteins and regulatory phosphatases are involved. The hope is that by studying how ERK is activated by different neurotransmitter systems and the ensuing downstream cellular modifications, the molecular basis of memory will be ultimately understood.     

Regulation of extracellular signal-regulated kinase phosphorylation in cultured rat striatal neurons

Recent studies demonstrate that activation of Ca²⁺-permeable N-methyl-D-aspartate (NMDA) receptors upregulates phosphorylation of mitogen-activated protein kinases (MAPKs) in heterologous cells and neurons. In cultured rat striatal neurons, the present work systematically evaluated the role of a number of protein kinases in forming a signaling cascade transducing NMDA receptor signals to MAPKs. It was found that a brief NMDA application consistently induced rapid and transient phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), a best characterized subclass of MAPKs. This ERK1/2 phosphorylation was resistant to the inhibition of protein kinase C, p38 MAPK, cyclin-dependent kinase 5, receptor tyrosine kinase (epidermal growth factor receptors), or non-receptor tyrosine kinases (including Src) by their selective inhibitors. However, the increase in ERK1/2 phosphorylation was partially blocked by a protein kinase A (PKA) inhibitor. The inhibitors for Ca²⁺/calmodulin-dependent protein kinase (CaMK) or phosphatidylinositol 3-kinase (PI3-kinase) completely blocked the NMDA-stimulated ERK1/2 phosphorylation. In an attempt to characterize the sequential role of CaMK and PI3-kinase, we found that NMDA increased PI3-kinase phosphorylation on Tyr⁵⁰⁸, which kinetically corresponded to the ERK1/2 phosphorylation and was blocked by the CaMK inhibitor. These results indicate that the protein kinases are differentially involved in linking NMDA receptors to ERK1/2 in striatal neurons.     

Role of extracellular signal-regulated kinase signal transduction pathway in anxiety

Extracellular signal-regulated kinase (ERK) signal transduction pathway is widely implicated in multiple physiological processes. However, it remains to be determined whether ERK pathway plays roles in anxiety. Here we investigated the changes of phosphorylated ERK1/2 (pERK1/2) and c-Fos expression by immunostaining in the medial prefrontal cortex (mPFC) of anxious rats. The results indicated that the levels of pERK and c-Fos were significantly increased during anxiety. Inhibition of ERK phosphorylation blocked the anxiety-induced c-Fos expression. In the animal behavioral tests, the PD98059-treated anxious rats had a significant increase in the numbers of the open-arm entries, the time spent in the open-arms and the numbers of head-dipping in EPM test, and increase the inner locomotion in the open field test compared with the anxious rats. The results suggested that the ERK signal transduction pathway might play an important role in anxiety, and inhibition of the ERK pathway in the mPFC could produce anxiolysis effect.     

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signaltransduction pathway in depressive disorder

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.     

Role of extracellular signal-regulated kinase signal transduction pathway in anxiety

Extracellular signal-regulated kinase (ERK) signal transduction pathway is widely implicated in multiple physiological processes. However, it remains to be determined whether ERK pathway plays roles in anxiety. Here we investigated the changes of phosphorylated ERK1/2 (pERK1/2) and c-Fos expression by immunostaining in the medial prefrontal cortex (mPFC) of anxious rats. The results indicated that the levels of pERK and c-Fos were significantly increased during anxiety. Inhibition of ERK phosphorylation blocked the anxiety-induced c-Fos expression. In the animal behavioral tests, the PD98059-treated anxious rats had a significant increase in the numbers of the open-arm entries, the time spent in the open-arms and the numbers of head-dipping in EPM test, and increase the inner locomotion in the open field test compared with the anxious rats. The results suggested that the ERK signal transduction pathway might play an important role in anxiety, and inhibition of the ERK pathway in the mPFC could produce anxiolysis effect.     

Asymmetric activation of extracellular signal-regulated kinase 1/2 in rat vestibular nuclei by unilateral labyrinthectomy

The expression of phosphorylated extracellular signal-regulated kinase 1/2 (pERK 1/2) was evaluated in the vestibular nuclei (VN) of rats following unilateral labyrinthectomy (UL). Immunohistochemistry revealed a significant asymmetrical increase in pERK 1/2 expression in the VN, 5 min after UL, after which pERK 1/2 immunoreactivity decreased rapidly and was undetectable by 90 min after UL. These results suggest that unilateral deafferentation of the vestibular system triggers intracellular signal pathways that activate ERK 1/2 in the VN.     

The extracellular signal-regulated kinase pathway contributes to the control of behavioral excitement

The extracellular signal-regulated kinase (ERK) pathway mediates neuronal plasticity in the CNS. The mood stabilizers lithium and valproate activate the ERK pathway in prefrontal cortex and hippocampus and potentiate ERK pathway-mediated neurite growth, neuronal survival and hippocampal neurogenesis. Here, we examined the role of the ERK pathway in behavioral plasticity related to facets of bipolar disorder. Mice with ERK1 ablation acquired reduced phosphorylation of RSK1, an ERK substrate, in prefrontal cortex and striatum, but not in hippocampus or cerebellum, indicating the ablation-induced brain region-specific ERK signaling deficits. ERK1 ablation produced a behavioral excitement profile similar to that induced by psychostimulants. The profile is characterized by hyperactivity, enhanced goal-directed activity and increased pleasure-related activity with potential harmful consequence. ERK1-ablated mice were hyperactive in multiple tests and resistant to behavioral despair in the forced swim test. These mice displayed more home-cage voluntary wheel running activities, rearings in a large arena and open-arm visits in an elevated plus maze. Treatments with valproate and olanzapine, but not lithium reduced baseline activities in ERK1-ablated mice. All three treatments attenuated amphetamine-induced hyperactivity in ablated mice. These data indicate a profound involvement of ERK1 signaling in behavioral excitement and in the behavioral action of antimanic agents. The extent to which ERK pathway perturbation contributes to the susceptibility, mood switch mechanism(s) and symptom pathophysiology of bipolar disorder requires further investigation. Whether there is a shared mechanism through which mood stabilizers produce their clinical actions on mood, thought and behavioral symptoms of mania also requires further investigation.     

Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2

Earlier studies identified the dynorphin-kappa opioid receptor (KOR) system as a critical mediator of dysphoria-induced aversion after repeated stress exposure, but the molecular signaling mechanisms were not fully characterized. In this study we report that repeated forced swim stress caused a significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 a mitogen-activated protein kinase (MAPK) in both the caudate and nucleus accumbens regions of the mouse striatum. Activation was blocked by the KOR antagonist, norbinaltorphimine, and absent in KOR knockout mice. In contrast to p38-MAPK activation by stress-induced dynorphin release, KOR-mediated ERK1/2 phosphorylation was not dependent on G-protein coupled receptor kinase 3 expression. These results indicate stress-induced activation of the dynorphin-KOR systems activates ERK1/2 MAPK signaling, and this may contribute to the behavioral responses to repeated stress exposure.     

Non-photic phase shifting of the circadian clock: role of the extracellular signal-responsive kinases I/II/mitogen-activated protein kinase pathway

The master circadian clock, located in the suprachiasmatic nucleus (SCN), is synchronized to the external world primarily through exposure to light. A second class of stimuli based on arousal or activity can also reset the hamster circadian clock in a manner distinct from light. The mechanism underlying these non-photic phase shifts is unknown, although suppression of canonical clock genes and immediate early genes has been implicated. Recently, suppression of one of the mitogen-activated protein kinases (MAPK), namely extracellular signal-responsive kinases I/II (ERK), has been implicated in phase shifts to dark pulses, a stimulus with both photic and non-photic components. We investigated the involvement of the ERK/MAPK pathway in phase shifts in response to 3 h of sleep deprivation initiated at mid-day. About three-quarters of animals subjected to this procedure demonstrated large phase advances of about 3 h. Those that shifted exhibited a significant decrease in phosphorylated ERK (p-ERK) in the SCN. Those animals that were perfused during the sleep deprivation also exhibited immunoreactivity for p-ERK in a distinct portion of the ventrolateral SCN. Finally, injections of U0126 to the SCN to prevent phosphorylation of ERK significantly decreased levels of p-ERK but did not produce phase shifts. These data demonstrate that a purely non-photic manipulation is able to alter the activity of the MAPK pathway in the SCN, with downregulation in the SCN shell and activation in a portion of the SCN core.     

Clusterin enhances proliferation of primary astrocytes through extracellular signal-regulated kinase activation

Clusterin, a secretory glycoprotein, has been shown to be up-regulated in the reactive astrocytes in response to brain injury and neurodegenerative diseases, but its function has not been clearly elucidated. In this study, we investigate whether clusterin has growth-stimulatory activity in astrocytes. Suppression of clusterin with antisense oligonucleotide induced growth arrest, whereas transient overexpression of clusterin by cDNA transfection or exogenous treatment with purified clusterin promoted proliferation of the primary astrocytes in culture. This clusterin-stimulated proliferation was abrogated by PD98059, an inhibitor of mitogen-activated protein kinase kinase. These results suggest that clusterin might play an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway.     

The role of the extracellular signal-regulated kinase pathway in cerebellar abnormalities in schizophrenia

Recent postmortem and functional imaging studies have revealed that cerebellar abnormalities may play a role in the pathophysiology of schizophrenia. Cerebellum is a part of the cortical-subcortical-cerebellar circuitry that is involved in higher cognitive functions. Deficits in cognition, including information, executive functions, attention, emotion, and memory have been described in patients with schizophrenia. Given the pivotal role of mitogen-activated protein (MAP) kinase pathways in regulation of neuronal function and especially the role of extracellular-signal regulated kinase (ERK) in synaptic plasticity, cell survival, learning and memory, the importance of MAP kinases in schizophrenia is being increasingly recognized. In this mini-review is summarized recent evidence from human postmortem studies and the phencyclidine (PCP) pharmacological model of schizophrenia that ERK signaling pathway could contribute to the pathogenic events that occur in the cerebellum in schizophrenia.     

Microglial signaling by amyloid beta protein through mitogen-activated protein kinase mediating phosphorylation of MARCKS

Myristoylated alanine-rich C kinase substrate (MARCKS), an acidic protein associated with cell motility and phagocytosis, is activated upon phosphorylation by protein kinase C (PKC) and proline-directed protein kinases. In Alzheimer disease (AD), activated microglia expressing MARCKS migrates around senile plaques. We reported that amyloid beta protein (A beta), a major component of senile plaques, activated MARCKS through a tyrosine kinase and PKC-delta. We have now identified another A beta signaling pathway through a mitogen-activated protein kinase (MAPK) involved in the phosphorylation of MARCKS and analysed cross-talk between PKC and MAPK pathways in primary cultured rat microglia. A selective inhibitor for MAPK kinase, PD098059, significantly inhibited the phosphorylation of MARCKS induced by A beta. Extracellulary regulated kinases, the activities of which were induced by A beta, directly phosphorylated a recombinant MARCKS in vitro. The MAPK pathway was sensitive to wortmannin, but not to a PKC inhibitor or to tyrosine kinase inhibitors. The activation of PKC by A beta was not sensitive to wortmannin. Our findings suggest involvement of the MAPK pathway through phosphoinositol 3-kinase in the phosphorylation of MARCKS in rat cultured microglia, an event may be associated with mechanisms activating microglia in AD.     

Sleep deprivation impairs spatial memory and decreases extracellular signal-regulated kinase phosphorylation in the hippocampus

Loss of sleep may result in memory impairment. However, little is known about the biochemical basis for memory deficits induced by sleep deprivation. Extracellular signal-regulated kinase (ERK) is involved in memory consolidation in different tasks. Phosphorylation of ERK is necessary for its activation and is an important step in mediating neuronal responses to synaptic activities. The aim of the present study was to determine the effects of total sleep deprivation (TSD) on memory and ERK phosphorylation in the brain. Rats were trained in Morris water maze to find a hidden platform (a spatial task) or a visible platform (a nonspatial task) after 6 h TSD or spontaneous sleep. TSD had no effect on spatial learning, but significantly impaired spatial memory tested 24 h after training. Nonspatial learning and memory were not impaired by TSD. Phospho-ERK levels in the hippocampus were significantly reduced after 6 h TSD compared to the controls and returned to the control levels after 2 h recovery sleep. Total ERK1 and ERK2 were slightly increased after 6 h TSD and returned to the control levels after 2 h recovery sleep. These alterations were not observed in the cortex after TSD. Protein phosphotase-1 and mitogen-activated protein kinase phosphatase-2, which dephosphorylates phospho-ERK, were also measured, but they were not altered by TSD. The impairments of both spatial memory and ERK phosphorylation indicate that the hippocampus is vulnerable to sleep loss. These results are consistent with the idea that decreased ERK activation in the hippocampus is involved in sleep deprivation-induced spatial memory impairment.     

Activation of extracellular signal-regulated kinase- mitogen-activated protein kinase cascade in the amygdala is required for memory reconsolidation of auditory fear conditioning

Consolidation of new fear memories has been shown to require de novo RNA and protein synthesis in the lateral nucleus of amygdala (LA). Recently we have demonstrated that consolidated fear memories, when reactivated, return to a labile state which is sensitive to disruption by the protein synthesis inhibitor anisomycin. The specific molecular mechanisms that underlie this reconsolidation of fear memories are still largely unknown. The activation of extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) pathway in the LA is required for the consolidation of auditory fear memories. In the present study, we examined the role of ERK-MAPK cascade in the LA during reconsolidation of auditory fear conditioning. We show that intra-LA infusions of the MAPK kinase (MEK) inhibitor U0126, a manipulation which inhibits activation of ERK-MAPK, impairs postreactivation long-term memory (PR-LTM) but leaves the postreactivation short-term memory (PR-STM) intact. The same treatment with U0126, in the absence of memory reactivation, has no effect. Furthermore, we verified that reconsolidation requires translation using a second protein synthesis inhibitor, cycloheximide. Post-reactivation infusions of cycloheximide blocked PR-LTM but not PR-STM and, in the absence of reactivation, had no effect. Our data show that activation of ERK-MAPK signalling pathway and protein synthesis in the LA are required for reconsolidation of auditory fear memories.     

D1 receptor regulation of preprotachykinin-A gene by extracellular signal-regulated kinase pathway in striatal cultures

In animal models of Parkinson's disease, a supersensitive response to dopamine (DA) is associated with a switch in the coupling of striatal DA D1 receptors from a cyclic AMP/protein kinase A signaling pathway to one involving extracellular signal-regulated kinase/mitogen-associated protein kinase. In this study, we found that generation of organotypic striatal cultures, with concomitant loss of DA innervation, led to a downregulation in preprotachykinin-A gene expression, which was reinstated by D1 receptor activation in an extracellular signal-regulated kinase/mitogen-associated protein kinase-dependent manner. These data demonstrate that acute organotypic slice cultures recapitulate important changes in D1 receptor-mediated signal transduction seen in DA-denervated animals, providing a valuable model system to study denervation effects on DA signaling and striatal gene expression.     

In vitro activation of extracellular signal-regulated kinase1/2 in the inner ear of guinea pigs

Growth factors such as vascular endothelial growth factor (VEGF) exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Although both VEGF and inactive ERK could be detected in the inner ear of guinea pigs, under normal conditions activated ERK (phospho-ERK) was found only sparely. Cochleae of adult guinea pigs were removed, incubated with VEGF in a carbogen-gased organ-bath for 5, 15, 30 and 60 min (n=6 in each group), fixed with PFA 4%, embedded in paraffin and sectioned, followed by immunohistochemical staining to inactive and active ERK. Whereas inactive ERK was found in all cochleae, in sensory and supporting cells of the apex activated ERK was strongly detected after 5-min VEGF-incubation. After 15 min all Corti-organs showed clear staining corresponding to activated ERK, which decreased again after 30 min. Faint staining in endothelial cells of the spring-coil-vessels and in the spiral ganglion cells was found after 30 min and was increased after 60 min, while the staining in the Corti-organs vanished. Addition of the MEK-inhibitor PD 98059 to the organ-bath led to diminished phospho-ERK1/2 immunostaining. These findings provide evidence for a VEGF-dependent phosphorylation of ERK1/2 in the cochlea. Activated ERK1/2 is thought to support axonal outgrowth, enhancement of cell survival and to regulate the turnover of the NO/cGMP-pathway.     

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.     

Olanzapine produces trophic effects in vitro and stimulates phosphorylation of Akt/PKB, ERK1/2, and the mitogen-activated protein kinase p38

Olanzapine has previously been shown to stimulate the growth of neuronal cells in culture. A major goal of the present studies was to determine if olanzapine also provided neuroprotection to pheochromocytoma (PC12) cells, SH-SY5Y neuroblastoma cells, and primary cultures of rat cortical neurons. Olanzapine was mitogenic and enhanced the survival of PC12 cells, SH-SY5Y cells and 3T3 preadipocytes, but not L6 myoblasts or myeloma cells. It protected neuronal cells from death induced by serum and glutamine deprivation, amyloid beta peptide (25-35), and fluphenazine. Molecular mechanisms of the neuroprotection by olanzapine were explored, specifically the activation of various protein kinase signaling pathways including Akt/protein kinase B (PKB), extracellular-regulated kinase (ERK), ERK1/2, and mitogen-activated protein kinase (MAPK), p38. Olanzapine treatment led to rapid phosphorylation of kinases from all three pathways in PC12 cells. Phosphorylation of Akt was blocked with selective inhibitors (wortmannin and LY294002), which implicates phosphoinositide 3-kinase (PI3K) in the signaling cascade. Short-term mitogenic effects of olanzapine were abolished with a selective inhibitor of Akt, but not by inhibition of the ERK pathway. Other antipsychotic drugs stimulated phosphorylation of a subset of the kinase panel, but not all three kinases. The present findings demonstrate that olanzapine has both mitogenic and neuroprotective effects in neuronal cells.