u-0126 and staurosporine-aglycone

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.

Topics: Animals; Benzylamines; Bicuculline; Brain-Derived Neurotrophic Factor; Butadienes; Calcium Signaling; Carbazoles; Cells, Cultured; Cerebral Cortex; Enzyme Induction; GABAergic Neurons; gamma-Aminobutyric Acid; Gene Expression Regulation; Glutamate Decarboxylase; Homeostasis; Indole Alkaloids; MAP Kinase Signaling System; Mice; Mice, Inbred ICR; Nitriles; Protein Isoforms; Protein Kinase Inhibitors; Receptor, trkB; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Signal Transduction; Sulfonamides

Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of BDNF, which were blocked by ERK and PI3K inhibitors, respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkB-ERK/PI3K-CREB signaling pathway in C6 glioma cells.

Topics: Animals; Brain-Derived Neurotrophic Factor; Butadienes; Carbazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; CREB-Binding Protein; Disaccharidases; Drugs, Chinese Herbal; Enzyme Inhibitors; Indole Alkaloids; MAP Kinase Signaling System; Models, Biological; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Plant Roots; Polygala; Rats; Receptor, trkB; Signal Transduction

Signaling pathways mediating Epstein-Barr virus (EBV) reactivation by Ag-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporine, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48 h after reactivation perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, glycogen synthase kinase 3β (GSK-3β), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK, respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication.

Topics: Animals; Antiviral Agents; Butadienes; Carbazoles; Cell Line, Tumor; Cell Survival; Genome, Viral; Herpesvirus 4, Human; Humans; Indole Alkaloids; MAP Kinase Signaling System; Microbial Sensitivity Tests; Mutagenesis, Site-Directed; NF-kappa B; Nitriles; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Sf9 Cells; Staurosporine; Viral Proteins; Virus Replication

We found that ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] caused phosphorylation of mitogen-activated protein kinase (MAPK), followed by expression of neurofilament-M, a neuron-specific protein, in cultured PC12 rat pheochromocytoma cells. The ebselen-induced MAPK activation was suppressed by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, a selective inhibitor of Trk family tyrosine kinases; AG1478, an antagonist of epidermal growth factor receptor (EGFR); pertussis toxin, an inhibitor of Gi/o; or GP antagonist-2A, an inhibitor of Gq. Furthermore, we observed that N-acetyl-L-cysteine, an inhibitor of tyrosine kinases, suppressed ebselen-induced MAPK activation and buthionine sulfoximine, an activator of protein tyrosine phosphatases, enhanced the effect, indicating that ebselen activated MEK1/2 through one or more tyrosine kinases. Based on these results, we propose that ebselen stimulated intracellular tyrosine kinase activity, thus activating a MAPK cascade (tyrosine kinase-MEK1/2-ERK1/2) in PC12 cells and that this activation resulted in their neuronal differentiation.

Topics: Animals; Antioxidants; Azoles; Butadienes; Carbazoles; Enzyme Activation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; Indole Alkaloids; Isoindoles; Mitogen-Activated Protein Kinases; Neurites; Neurofilament Proteins; Nitriles; Organoselenium Compounds; Oxidation-Reduction; PC12 Cells; Pertussis Toxin; Phosphorylation; Quinazolines; Rats; Receptor Protein-Tyrosine Kinases; Selenium; Tyrphostins

N-Methyl-D-aspartate (NMDA) at a subtoxic concentration (100 microM) promotes neuronal survival against glutamate-mediated excitotoxicity via a brain-derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3-K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration- and time-dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK-801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 microM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3-K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl-2, bcl-X(L), and bax mRNA levels,. NMDA increases Bcl-2 and Bcl-X(L) protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate-mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl-2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.

Topics: Animals; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Butadienes; Carbazoles; Chromones; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Glutamic Acid; Indole Alkaloids; Mitogen-Activated Protein Kinase Kinases; Morpholines; N-Methylaspartate; Neurons; Neuroprotective Agents; Nitriles; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptor, trkB; RNA, Messenger; Signal Transduction

Inflammation of the primary afferent proximal to the dorsal root ganglion (DRG) and the DRG itself is known to produce radicular pain. Here, we examined pain-related behaviors and the activation of extracellular signal-regulated protein kinase (ERK) in the DRG after inflammation near the DRG somata. Inflammation of the L4/5 nerve roots and DRG induced by complete Freund's adjuvant (CFA) produced mechanical allodynia on the ipsilateral hindpaw and induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase (trk) A-expressing small- and medium-size neurons. This CFA-induced increase in ERK phosphorylation was mediated through trk receptors, because intrathecal treatment with the tyrosine kinase inhibitor, K252a, reduced the activation of ERK. On the other hand, an increase in brain-derived neurotrophic factor (BDNF) mRNA/protein in the DRG concomitant with the ERK activation was also observed. Furthermore, we found that nerve growth factor (NGF) injection directly into the L4/5 nerve roots and DRG produced mechanical allodynia, and an increase in the phosphorylation of ERK and BDNF expression in the DRG, but the mitogen-activated protein kinase (MAPK) kinase1/2 inhibitor, U0126, inhibited the effects induced by NGF. Therefore, we suggest that after inflammation near the cell body, NGF synthesized within the nerve root and DRG induces BDNF expression through trkA receptors and intracellular ERK-MAPK. The activation of MAPK in the primary afferents may be involved in the pathophysiological mechanisms of inflammation-induced radiculopathy and MAPK pathways in the primary afferents may be potential targets for pharmacological intervention for neuropathic pain produced by inflammation near the DRG somata.

Topics: Animals; Behavior, Animal; Blotting, Western; Brain-Derived Neurotrophic Factor; Butadienes; Carbazoles; Cell Count; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Indole Alkaloids; Male; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Neurons; Nitriles; Pain; Pain Measurement; Phosphorylation; Radiculopathy; Rats; Rats, Sprague-Dawley; Reaction Time; Spinal Nerve Roots; Time Factors

Brain-derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival-promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death-enhancing effect of BDNF remains unknown, in contrast to that of its survival-promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor-induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF-accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB-IgG and K252a blocked the BDNF-induced acceleration of the death, indicating that the death-accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF-accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen-activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF-accelerated, NO donor-induced apoptosis.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Butadienes; Carbazoles; Cell Nucleus; Cell Survival; Cells, Cultured; Cerebral Cortex; Cycloheximide; Dactinomycin; DNA Fragmentation; Drug Synergism; Embryo, Mammalian; Enzyme Inhibitors; Female; Imidazoles; Immunoglobulin G; Indole Alkaloids; Kinetics; Male; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurons; Nitric Oxide Donors; Nitriles; Nitroprusside; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Wistar; Receptor, trkB; Signal Transduction