ABSTRACT
BACKGROUND: It is uncertain how electroconvulsive therapy-induced generalized seizures exert their potent therapeutic effects on various neuropsychiatric disorders. Adenosine monophosphate-activated protein kinase (AMPK) plays a major role in maintaining metabolic homeostasis and activates autophagic processes via unc-51-like kinase (ULK1). Evidence supports the involvement of autophagy system in the action mechanisms of antidepressants and antipsychotics. The effect of electroconvulsive therapy on autophagy-related signaling requires further clarification. METHODS: The effect of electroconvulsive seizure on autophagy and its association with the AMPK signaling pathway were investigated in the rat frontal cortex. Electroconvulsive seizure was provided once per day for 10 days (E10X), and compound C or 3-methyadenine was administered through an intracerebroventricular cannula. Molecular changes were analyzed with immunoblot, immunohistochemistry, and transmission electron microscopy analyses. RESULTS: E10X increased p-Thr172-AMPKα immunoreactivity in rat frontal cortex neurons. E10X increased phosphorylation of upstream effectors of AMPK, such as LKB1, CaMKK, and TAK1, and of its substrates, ACC, HMGR, and GABABR2. E10X also increased p-Ser317-ULK1 immunoreactivity. At the same time, LC3-II and ATG5-ATG12 conjugate immunoreactivity increased, indicating activation of autophagy. An intracerebroventricular injection of the AMPK inhibitor compound C attenuated the electroconvulsive seizure-induced increase in ULK1 phosphorylation as well as the protein levels of LC3-II and Atg5-Atg12 conjugate. Transmission electron microscopy clearly showed an increased number of autophagosomes in the rat frontal cortex after E10X, which was reduced by intracerebroventricular treatment with the autophagy inhibitor 3-methyadenine and compound C. CONCLUSIONS: Repeated electroconvulsive seizure treatments activated in vivo autophagy in the rat frontal cortex through the AMPK signaling pathway.
Subject(s)
Autophagosomes , Autophagy/physiology , Electroconvulsive Therapy , Frontal Lobe/physiology , Protein Kinases/metabolism , Seizures/metabolism , Signal Transduction/physiology , AMP-Activated Protein Kinase Kinases , Animals , Disease Models, Animal , Frontal Lobe/cytology , Frontal Lobe/diagnostic imaging , Frontal Lobe/metabolism , Male , Rats , Rats, Sprague-DawleyABSTRACT
Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.
Subject(s)
Depression/therapy , Electroshock/methods , Intramolecular Oxidoreductases/pharmacology , Macrophage Migration-Inhibitory Factors/pharmacology , Motor Activity/physiology , Analysis of Variance , Animals , Blotting, Western , Brain-Derived Neurotrophic Factor/metabolism , Cells, Cultured , DNA Primers/genetics , Immunohistochemistry , Infusions, Intraventricular , Intramolecular Oxidoreductases/administration & dosage , Intramolecular Oxidoreductases/genetics , Macrophage Migration-Inhibitory Factors/administration & dosage , Macrophage Migration-Inhibitory Factors/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/drug effects , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Serotonin/metabolism , Tryptophan Hydroxylase/metabolismABSTRACT
A growing body of evidence suggests that the circadian molecular system is involved in the pathogenic and therapeutic mechanisms underlying bipolar disorders. Lithium, a representative mood stabilizer, has been reported to induce the Period 2 (PER2) gene; however, the underlying molecular mechanisms require further study. We found that lithium upregulated PER2 expression at the transcriptional level in neuronally differentiated SH-SY5Y human neuroblastoma cells. Promoter reporter analyses using serial deletions of the PER2 promoter revealed that two early growth response 1 (Egr1)-binding sites (EBS) between positions -180 and -100 are required for maximal activation of the PER2 promoter by lithium. Ectopic expression of Egr1 enhanced lithium-induced PER2 promoter activity, while a point mutation in EBS abolished it. Electrophoretic mobility shift assays and chromatin immunoprecipitation indicated that Egr1 bound directly to the PER2 promoter. Stimulation of the extracellular-signal regulated kinase (ERK)1/2/Elk1 pathway by lithium was functionally linked to PER2 expression through Egr1 induction, and lithium-induced PER2 expression was strongly attenuated by depletion of Egr1 by siRNA. Lithium also upregulated the expression of Per2 and Egr1 in mouse frontal cortex. Induction of Per2 by lithium was attenuated in Egr1(-/-) mice. In conclusion, lithium stimulates PER2 transcription through the ERK/Elk1/Egr1 pathway in neuronal cells, indicating a connection between the ERK-Egr1 pathway and a circadian gene system in the mechanism of action of lithium.
Subject(s)
Early Growth Response Protein 1/physiology , Gene Expression Regulation , Lithium/pharmacology , Neuroblastoma/genetics , Period Circadian Proteins/genetics , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Blotting, Northern , Blotting, Western , Chromatin Immunoprecipitation , Early Growth Response Protein 1/antagonists & inhibitors , Electrophoretic Mobility Shift Assay , Fluorescent Antibody Technique , Humans , Luciferases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , Period Circadian Proteins/antagonists & inhibitors , Period Circadian Proteins/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation , Tumor Cells, Cultured , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolismABSTRACT
The enzymatic activity of histone deacetylases (HDACs) leads to a histone deacetylation-mediated condensed chromatic structure, resulting in transcriptional repression, which has been implicated in the modifications of neural circuits and behaviors. Repeated treatment with electroconvulsive seizure (ECS) induces changes in histone acetylation, expression of various genes, and intrabrain cellular changes, including neurogenesis. In this study, we examined the effects of repeated ECS on the expression of class I HDACs and related changes in histone modifications and gene expression in the rat frontal cortex. Ten days of repeated ECS treatments (E10X) up-regulated HDAC2 expression at the mRNA and protein levels in the rat frontal cortex compared with sham-treated controls; this was evident in the nuclei of neuronal cells in the prefrontal, cingulate, orbital, and insular cortices. Among the known HDAC2 target genes, mRNA expression of N-methyl-d-aspartate (NMDA) receptor signaling-related genes, including early growth response-1 (Egr1), c-Fos, glutamate receptor, ionotropic, N-methyl d-aspartate 2A (Nr2a), Nr2b, neuritin1 (Nrn1), and calcium/calmodulin-dependent protein kinase II alpha (Camk2α), were decreased, and the histone acetylation of H3 and/or H4 proteins was also reduced by E10X. Chromatin immunoprecipitation analysis revealed that HDAC2 occupancy in the promoters of down-regulated genes was increased significantly. Moreover, administration of sodium butyrate, a HDAC inhibitor, during the course of E10X ameliorated the ECS-induced down-regulation of genes in the rat frontal cortex. These findings suggest that induction of HDAC2 by repeated ECS treatment could play an important role in the down-regulation of NMDA receptor signaling-related genes in the rat frontal cortex through histone modification.
Subject(s)
Electroshock/adverse effects , Frontal Lobe/enzymology , Gene Expression Regulation/physiology , Histone Deacetylase 2/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Seizures , Acetylation/drug effects , Analysis of Variance , Animals , Butyric Acid/therapeutic use , Chromatin Immunoprecipitation , Disease Models, Animal , Frontal Lobe/drug effects , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Histamine Antagonists/therapeutic use , Male , Phosphopyruvate Hydratase/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Seizures/drug therapy , Seizures/enzymology , Seizures/etiology , Signal Transduction/drug effectsABSTRACT
RATIONALE: Electroconvulsive therapy (ECT) is an effective treatment modality for schizophrenia. However, its antipsychotic-like mechanism remains unclear. OBJECTIVES: To gain insight into the antipsychotic-like actions of ECT, this study investigated how repeated treatments of electroconvulsive seizure (ECS), an animal model for ECT, affect the behavioral and transcriptomic profile of a neurodevelopmental animal model of schizophrenia. METHODS: Two injections of MK-801 or saline were administered to rats on postnatal day 7 (PN7), and either repeated ECS treatments (E10X) or sham shock was conducted daily from PN50 to PN59. Ultimately, the rats were divided into vehicle/sham (V/S), MK-801/sham (M/S), vehicle/ECS (V/E), and MK-801/ECS (M/E) groups. On PN59, prepulse inhibition and locomotor activity were tested. Prefrontal cortex transcriptomes were analyzed with mRNA sequencing and network and pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) analyses were subsequently conducted. RESULTS: Prepulse inhibition deficit was induced by MK-801 and normalized by E10X. In M/S vs. M/E model, Egr1, Mmp9, and S100a6 were identified as center genes, and interleukin-17 (IL-17), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF) signaling pathways were identified as the three most relevant pathways. In the V/E vs. V/S model, mitophagy, NF-κB, and receptor for advanced glycation end products (RAGE) pathways were identified. qPCR analyses demonstrated that Igfbp6, Btf3, Cox6a2, and H2az1 were downregulated in M/S and upregulated in M/E. CONCLUSIONS: E10X reverses the behavioral changes induced by MK-801 and produces transcriptional changes in inflammatory, insulin, and mitophagy pathways, which provide mechanistic insight into the antipsychotic-like mechanism of ECT.
Subject(s)
Antipsychotic Agents , Electroconvulsive Therapy , Schizophrenia , Rats , Animals , Dizocilpine Maleate/pharmacology , NF-kappa B , Schizophrenia/chemically induced , Schizophrenia/therapy , Antipsychotic Agents/pharmacology , Seizures/chemically induced , Seizures/metabolismABSTRACT
OBJECTIVE: Electroconvulsive seizure (ECS) is a potent treatment modality for various neuropsychiatric diseases, including Parkinson disease (PD). Recent animal studies showed that repeated ECS activates autophagy signaling, the impairment of which is known to be involved in PD. However, the effectiveness of ECS on PD and its therapeutic mechanisms have not yet been investigated in detail. METHODS: Systemic injection of a neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), which destroys dopaminergic neurons in the substantia nigra compacta (SNc), in mice was utilized to induce an animal model of PD. Mice were treated with ECS 3 times per week for 2 weeks. Behavioral changes were measured with a rotarod test. Molecular changes related to autophagy signaling in midbrain including SNc, striatum, and prefrontal cortex were analyzed with immunohistochemistry and immunoblot analyses. RESULTS: Repeated ECS treatments normalized the motor deficits and the loss of dopamiergic neurons in SNc of the MPTP PD mouse model. In the mouse model, LC3-II, an autophagy marker, was increased in midbrain while decreased in prefrontal cortex, both of which were reversed by repeated ECS treatments. In the prefrontal cortex, ECS-induced LC3-II increase was accompanied with AMP-activated protein kinase (AMPK)-Unc-51-like kinase 1-Beclin1 pathway activation and inhibition of mamalian target of rapamycin signaling which promotes autophagy initiation. CONCLUSION: The findings revealed the therapeutic effects of repeated ECS treatments on PD, which could be attributed to the neuroprotective effect of ECS mediated by AMPK-autophagy signaling.
ABSTRACT
Clozapine is an antipsychotic drug that has a greater efficacy than other medications in some contexts, especially for the treatment of treatment-resistant schizophrenia. However, clozapine induces more metabolic side-effects involving abnormality in lipid metabolism compared to other antipsychotics. AMP-activated protein kinase (AMPK) plays a central role in controlling lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT1) pathway. In this study, we investigated the effect of a single intraperitoneal injection of clozapine on the AMPK-ACC-CPT1 pathway in the rat frontal cortex, which has been implicated as a target site for this antipsychotic drug. At 2 h after injection, the clinically relevant dose of clozapine had activated AMPK, with increased phosphorylation of AMPKα at Thr(172), and had inactivated ACC, with increased phosphorylation of ACC at Ser(79). In addition, clozapine activated the brain-specific isoform of CPT1, CPT1c, whose activity is inhibited by unphosphorylated ACC, in the rat frontal cortex. Immunohistochemistry and immunofluorescence analysis showed that clozapine induced an increase in number of p-AMPKα (Thr(172))- and p-ACC (Ser(79))-positive cells among the neurons of the rat frontal cortex. Taken together, these results show that clozapine activated the AMPK-ACC-CPT1 pathway in the neurons of the rat frontal cortex. These findings indicate that the antipsychotic agent clozapine affects the lipid regulatory system of neurons in the brain.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/metabolism , Antipsychotic Agents/pharmacology , Carnitine O-Palmitoyltransferase/metabolism , Clozapine/pharmacology , Frontal Lobe/drug effects , Signal Transduction/drug effects , Analysis of Variance , Animals , Dose-Response Relationship, Drug , Frontal Lobe/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Male , Phosphorylation/drug effects , Phosphorylation/physiology , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
RATIONALE: Accumulating evidence indicates critical involvement of mammalian target of rapamycin (mTOR) in the treatment of depressive disorders, epilepsy, and neurodegenerative disorders through its signal transduction mechanisms related to protein translation, autophagy, and synaptic remodeling. Electroconvulsive seizure (ECS) treatment is a potent antidepressive, anti-convulsive, and neuroprotective therapeutic modality; however, its effects on mTOR signaling have not yet been clarified. METHODS: The effect of ECS on the mTOR complex 1 (mTORC1) pathway was investigated in the rat frontal cortex. ECS or sham treatment was administered once per day for 10 days (E10X or sham), and compound C was administered through the intracerebroventricular cannula. Changes in mTORC1-associated signaling molecules and their interactions were analyzed. RESULTS: E10X reduced phosphorylation of mTOR downstream substrates, including p70S6K, S6, and 4E-BP1, and increased inhibitory phosphorylation of mTOR at Thr2446 compared to the sham group in the rat frontal cortex, indicating E10X-induced inhibition of mTORC1 activity. Akt and ERK1/2, upstream kinases that activate mTORC1, were not inhibited; however, AMPK, which can inhibit mTORC1, was activated. AMPK-responsive phosphorylation of Raptor at Ser792 and TSC2 at Ser1387 inhibiting mTORC1 was increased by E10X. Moreover, intrabrain inhibition of AMPK restored E10X-induced changes in the phosphorylation of S6, Raptor, and TSC2, indicating mediation of AMPK in E10X-induced mTOR inhibition. CONCLUSIONS: Repeated ECS treatments inhibit mTORC1 signaling by interactive crosstalk between mTOR and AMPK pathways, which could play important roles in the action of ECS via autophagy induction.
Subject(s)
AMP-Activated Protein Kinases , TOR Serine-Threonine Kinases , AMP-Activated Protein Kinases/metabolism , Animals , Frontal Lobe/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphorylation , Rats , Seizures , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induced behavioral changes in rats, a putative animal model for bipolar disorder. The binding of ouabain to Na-K ATPase is known to affect signaling molecules in vitro such as extracellular signal-regulated kinase1/2 (ERK1/2). Although ERK has been suggested to be related to the behavioral alterations induced by various psychotomimetics, the effect of ouabain on ERK in the brain related to behavioral changes has not been examined. After ICV injection of ouabain in rats, we investigated changes in the phosphorylation of mitogen-activated protein kinase kinase1/2 (MEK1/2), ERK1/2, and p90 ribosomal s6 kinase (p90RSK) in rat striatum, frontal cortex, and hippocampus along with changes in locomotor activity. Ouabain induced the following biphasic dose-dependent changes in locomotor activity: no change with 10(-6) M, a statistically significant decrease with 10(-5) M, no change with 10(-4) M, and a statistically significant increase with 0.5x10(-3) and 10(-3) M. The phosphorylation level of MEK1/2, ERK1/2, and p90RSK in rat striatum showed dose-dependent changes similar to those observed in locomotor activity with relatively high correlation. The phosphorylation of these molecules in rat frontal cortex and hippocampus also changed in a similar dose-dependent pattern. Taken together, ouabain induced biphasic dose-dependent changes in locomotor activity and the phosphorylation of the ERK1/2 pathway. These findings suggest a possible relationship between ouabain-induced behavioral changes and ERK activity in the brain and suggest an important role of ERK in regulating locomotor activity and mood state.
Subject(s)
Brain/enzymology , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Motor Activity/drug effects , Ouabain/pharmacology , Signal Transduction/drug effects , Animals , Behavior, Animal/drug effects , Brain/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic/drug effects , Injections, Intraventricular/methods , Male , Phosphorylation/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
Clozapine, a representative atypical antipsychotic, has superior efficacy compared to other antipsychotic agents and is used for the treatment of severe psychotic disorders. Therefore, studies on its mechanisms of action are important for understanding the mechanisms of therapeutic approaches to psychosis. Adenosine monophosphate-activated protein kinase (AMPK) is a serine-threonine kinase that plays a major role in maintaining metabolic homeostasis. Unc-51-like kinase 1 (ULK1) and Beclin1 are downstream substrates of AMPK and activate the autophagic process. In this study, we examined the effects of clozapine on the AMPK-ULK1-Beclin1 signaling pathway and autophagy in the frontal cortex of the rat. Clozapine (10mg/kg) administration increased the immunoreactivity of p-Thr172-AMPKα in the rat frontal cortex at 1, 2, and 4h after injection, as we previously reported. The immunoreactivity of p-Ser317-ULK1 and p-Ser93-Beclin1 was also increased at 2 and 4h after clozapine injection. At the same time, the immunoreactivity of LC3-II and the Atg5-Atg12 conjugate, which indicate activation of autophagy, was increased. Transmission electron microscopy clearly showed an increase in autophagosome number in the rat frontal cortex at 2h after clozapine injection. To investigate the role of AMPK in clozapine-induced autophagy, the effects of intracerebroventricular injection of compound C, an AMPK inhibitor, were examined. Administration of compound C attenuated the clozapine-induced increase in ULK1 and Beclin1 phosphorylation, as well the protein levels of LC3-II and the Atg5-Atg12 conjugate in the frontal cortex. In summary, the results showed that clozapine activates autophagy through the AMPK-ULK1-Beclin1 signaling pathway in the frontal cortex of the rat.
Subject(s)
Antipsychotic Agents/pharmacology , Autophagy/drug effects , Clozapine/pharmacology , Frontal Lobe/drug effects , Adenylate Kinase/metabolism , Animals , Autophagy/physiology , Autophagy-Related Protein-1 Homolog/metabolism , Beclin-1/metabolism , Frontal Lobe/metabolism , Frontal Lobe/ultrastructure , Male , Microscopy, Electron, Transmission , Phosphorylation/drug effects , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Signal Transduction/drug effectsABSTRACT
An elevated plus maze (EPM) test was used to determine if the 5-HT1A, GABAA, and benzodiazepine receptors play a role in the anxiolytic-like effects of a 50% EtOH extract of Cinnamomum cassia (C. cassia) in mice. A single treatment with C. cassia (750 mg/kg, p.o.) significantly increased the number of entries into and the time spent in the open arms of the EPM compared with the controls. A repeated treatment with C. cassia (100 mg/kg, 5 days, p.o.) significantly increased the time spent in the open arms of the EPM. Moreover, WAY 100635, (+)-bicuculline, and flumazenil blocked the effect of C. cassia. However, there were no changes in the locomotor activity and horizontal wire test observed in any group compared with the controls. Taken together, these results show that C. cassia has no adverse effects, such as myorelaxant effects, and might be an effective anxiolytic agent by regulating the serotonergic and GABAergic system.
Subject(s)
Anti-Anxiety Agents , Cinnamomum/chemistry , Receptor, Serotonin, 5-HT1A/drug effects , Receptors, GABA-A/drug effects , Animals , Bicuculline/pharmacology , Ethanol , Flumazenil/pharmacology , GABA Antagonists/pharmacology , GABA Modulators , Male , Mice , Mice, Inbred ICR , Motor Activity/drug effects , Muscle Relaxants, Central , Piperazines/pharmacology , Plant Extracts/pharmacology , Postural Balance/drug effects , Pyridines/pharmacology , Serotonin Antagonists/pharmacology , SolventsABSTRACT
Intracerebroventricular (ICV) injection of ouabain, a specific Na/K-ATPase inhibitor, induces behavioral changes in rats in a putative animal model of mania. The binding of ouabain to Na/K-ATPase affects signaling molecules in vitro, including ERK1/2 and Akt, which promote protein translation. We have also reported that ERK1/2 and Akt in the brain are involved in the ouabain-induced hyperactivity of rats. In this study, rats were given an ICV injection of ouabain, and then their frontal cortices were examined to determine the effects of ouabain on the mTOR/p70S6K/S6 signaling pathway and protein translation, which are important in modifications of neural circuits and behavior. Rats showed ouabain-induced hyperactivity up to 8h following injection, and increased phosphorylation levels of mTOR, p70S6K, S6, eIF4B, and 4E-BP at 1, 2, 4, and 8h following ouabain injection. Immunohistochemical analyses revealed that increased p-S6 immunoreactivity in the cytoplasm of neurons by ouabain was evident in the prefrontal, cingulate, and orbital cortex. These findings suggested increased translation initiation in response to ouabain. The rate of protein synthesis was measured as the amount of [(3)H]-leucine incorporation in the cell-free extracts of frontal cortical tissues, and showed a significant increase at 8h after ouabain injection. These results suggest that ICV injection of ouabain induced activation of the protein translation initiation pathway regulated by ERK1/2 and Akt, and prolonged hyperactivity in rats. In conclusion, protein translation pathway could play an important role in ouabain-induced hyperactivity in a rodent model of mania.
Subject(s)
Enzyme Inhibitors/pharmacology , Frontal Lobe/drug effects , Ouabain/pharmacology , Protein Biosynthesis/drug effects , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Animals , Carrier Proteins/metabolism , Enzyme Inhibitors/administration & dosage , Eukaryotic Initiation Factors/metabolism , Frontal Lobe/metabolism , Injections, Intraventricular , Intracellular Signaling Peptides and Proteins , Male , Motor Activity/drug effects , Ouabain/administration & dosage , Phosphoproteins/metabolism , Phosphorylation/drug effects , Rats , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Ribosome Subunits, Small/metabolismABSTRACT
RATIONALE: Clozapine affects the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the brain, which plays an important role in its antipsychotic action. However, previous findings are inconsistent, and related molecular mechanisms require further clarification. OBJECTIVES: Time- and dose-dependent effects of clozapine on the ERK1/2 pathway and its regulatory mechanism were investigated in rat frontal cortex. METHODS AND RESULTS: At 15, 30, 60, and 120 min after intraperitoneal injection of clozapine (5, 10, and 20 mg/kg), changes in ERK1/2, its upstream canonical kinases (Raf1 and mitogen-activated protein kinase kinase 1/2 [MEK1/2]), and its downstream molecule (p90 ribosomal S6 kinase [p90RSK]) were investigated in rat frontal cortex. At 15 min, p-Raf1, p-MEK1/2, p-ERK1/2, and p-p90RSK all increased dose-dependently. At 30 min, p-ERK1/2 and p-p90RSK showed no significant changes, while dose-dependent increases in p-Raf1 and p-MEK1/2 were found. At 60 and 120 min, although p-ERK1/2 and p-p90RSK decreased, increases in p-Raf1 and p-MEK1/2 were maintained. A clozapine-induced reduction in ERK1/2 phosphorylation was evident at both tyrosine and threonine residues, suggesting the involvement of dual specificity phosphatases (DUSPs; mitogen-activated protein kinase phosphatases [MKPs]). mRNA expression of seven Dusps that can dephosphorylate ERK1/2 were examined; Mkp-1 (Dusp1) mRNA increased following clozapine treatment. Moreover, MKP-1 protein and phosphatase activity increased, and binding of MKP-1 to ERK1/2 was also upregulated by clozapine administration. CONCLUSIONS: In rat frontal cortex, clozapine regulates ERK1/2 phosphorylation via MKP-1, which induces uncoupling between Raf1-MEK1/2 and ERK1/2-p90RSK activity. These findings suggest an important role of MKP-1 in the mechanism of action of clozapine.
Subject(s)
Antipsychotic Agents/pharmacology , Clozapine/pharmacology , Dual Specificity Phosphatase 1/metabolism , Animals , Antipsychotic Agents/administration & dosage , Clozapine/administration & dosage , Dose-Response Relationship, Drug , Frontal Lobe/drug effects , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , MAP Kinase Kinase Kinases/metabolism , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Proto-Oncogene Proteins c-raf , Rats , Rats, Sprague-Dawley , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction/drug effects , Time FactorsABSTRACT
Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000µM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000µM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000µM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model.
Subject(s)
Corpus Striatum/drug effects , Corpus Striatum/enzymology , Enzyme Inhibitors/administration & dosage , Mitogen-Activated Protein Kinase 3/metabolism , Ouabain/administration & dosage , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Tyrosine 3-Monooxygenase/metabolism , Animals , Disease Models, Animal , Enzyme Activation/drug effects , Enzyme Activation/physiology , Injections, Intraventricular , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Sodium-Potassium-Exchanging ATPase/physiologyABSTRACT
Cyclosporin A (CsA) is an inhibitor of calcineurin, a calcium/calmodulin dependent serine/threonine phosphatase. Protein kinase C (PKC) is a family of serine/threonine kinases. Both calcineurin and PKC are implicated in psychiatric diseases and the therapeutic mechanisms of treatment agents. It has been reported that calcineurin interacts with components of PKC signaling pathways. We administrated 50mg/kg CsA into rats by intraperitoneal injection and examined the acute effect of single systemic CsA on the locomotor activity of rats and the phosphorylation of PKC and its substrates GAP43 and MARCKS. Systemic CsA increased locomotor activity beginning 1h after injection. The immunoreactivity of p-MARCKS(S152/156) was higher in the CsA group 1h after injection, whereas p-GAP43(S41) immunoreactivity was increased by CsA after 5h. The immunoreactivity of p-PKC pan was increased by CsA at both 1 and 5h after administration. Our data suggest that activation of the PKC pathway might be related to CsA-induced hyperlocomotion.
Subject(s)
Cyclosporine/pharmacology , GAP-43 Protein/metabolism , Hippocampus/drug effects , Immunosuppressive Agents/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Signal Transduction/drug effects , Animals , Blotting, Western , Calcineurin/metabolism , Hippocampus/metabolism , Male , Motor Activity/drug effects , Myristoylated Alanine-Rich C Kinase Substrate , Phosphorylation/drug effects , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
Cyclosporine A (CsA), an immunosuppressant and calcineurin inhibitor, induces hyperlipidemia in humans and animals. AMP-activated protein kinase (AMPK) is involved in metabolic homeostasis and lipid metabolism through modulating downstream molecules acetyl CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). AMPK activity is regulated by the phosphorylation at the Thr-172 residue by its upstream liver kinase B 1 (LKB1), Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) or transforming growth-factor-ß-activated kinase 1 (TAK1). AMPK can be deactivated through dephosphorylation by protein phosphatase 2Cα (PP2Cα). In this study, we demonstrated that phosphorylation at Thr-172-AMPK increased with a concurrent increase in the phosphorylation of Ser-431-LKB1 and Thr-184/187-TAK1 in the rat hippocampus at 5 h after an intraperitoneal CsA (50 mg/kg) injection. CsA did not affect the phosphorylation of Thr-196-Ca(2+)/calmodulin-dependent protein kinase 4 (CaMK4) and the amount of PP2Cα. An increased phosphorylation of Ser-79-ACC and Ser-872-HMG-CoAR was also observed. In conclusion, our data indicate that CsA activates the AMPK pathway in the rat hippocampus, which suggests that CsA affects the regulatory signaling pathway of lipid metabolism in the rat brain.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cyclosporine/pharmacology , Enzyme Inhibitors/pharmacology , Hippocampus/drug effects , Signal Transduction/drug effects , AMP-Activated Protein Kinase Kinases , Animals , Hippocampus/metabolism , Lipid Metabolism/drug effects , MAP Kinase Kinase Kinases/metabolism , Male , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na-K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1mM of ouabain. Phosphorylation of GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8h after a single 1mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania.
Subject(s)
Brain/drug effects , Motor Activity/drug effects , Ouabain/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Analysis of Variance , Animals , Blotting, Western , Brain/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Forkhead Transcription Factors/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Injections, Intraventricular , Male , Nerve Tissue Proteins/metabolism , Nitric Oxide Synthase Type III/metabolism , Phosphorylation/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
Protein kinase C (PKC) has been suggested as a molecular target related to the pathogenetic and therapeutic mechanisms of mood disorders in which electroconvulsive seizure (ECS) is effective. However, the reports concerning the effects of ECS on PKC are anecdotal and need further clarification. In this study, we examined the effects of ECS treatment on the phosphorylation of PKC substrates, including GAP-43, MARCKS, and neurogranin. Immunoblot using anti-p-PKC substrate antibodies revealed that a single ECS treatment induced temporal changes in the phosphorylation level of PKC substrates in rat brain, reflecting the effects on PKC activity. Phosphorylation of GAP-43 and MARCKS, representative PKC substrates related to synaptic remodeling, increased from 5 to 30 min, after a transient decrease at 0 min immediately after ECS, and returned to basal levels at 60 min in rat frontal cortex, hippocampus, and cerebellum. Phosphorylation of neurogranin, another PKC substrate, showed a similar pattern of temporal changes in the frontal cortex and hippocampus. Immunohistochemical analysis revealed that p-GAP-43 and p-MARCKS were densely stained throughout the neuronal cells of the prefrontal cortex and hippocampus, and the Purkinje cells of cerebellum, after ECS treatment. Brief and transient activation of PKC may be translated into long-term biochemical changes, resulting in synaptic plasticity. Taken together, the acute effects of ECS on PKC activity, which could be an underpinning of long-term biochemical changes induced by ECS, may contribute to understand the molecular mechanism of ECS.
Subject(s)
Brain/metabolism , GAP-43 Protein/metabolism , Gene Expression Regulation/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neurogranin/metabolism , Seizures/pathology , Analysis of Variance , Animals , Disease Models, Animal , Electroshock/adverse effects , Male , Myristoylated Alanine-Rich C Kinase Substrate , Phosphorylation , Rats , Rats, Sprague-Dawley , Seizures/etiology , Seizures/metabolismABSTRACT
Haloperidol, a classical antipsychotic drug, affects the extracellular signal-regulated kinase (ERK) pathway in the brain. However, findings are inconsistent and the mechanism by which haloperidol regulates ERK is poorly understood. Therefore, we examined the ERK pathway and the related protein phosphatase 2A (PP2A) in detail after haloperidol administration. Haloperidol (0.5 and 1 mg/kg) induced biphasic changes in the phosphorylation level of mitogen-activated protein kinase kinase (MEK), ERK, and p90 ribosomal S6 kinase (p90RSK) without changing Raf-1 phosphorylation. Fifteen minutes after haloperidol administration, MEK-ERK-p90RSK phosphorylation increased, whilst PP2A activity decreased. At 60 min, the reverse was observed and the binding of PP2A to MEK and ERK increased. Higher dosages of haloperidol (2 and 4 mg/kg), affected neither MEK-ERK-p90RSK phosphorylation nor PP2A activity. Accordingly, PP2A regulates acute dose- and time-dependent changes in MEK-ERK-p90RSK phosphorylation after haloperidol treatment. These findings suggest the involvement of a dephosphorylating mechanism in the acute action of haloperidol.
Subject(s)
Antipsychotic Agents/pharmacology , Extracellular Signal-Regulated MAP Kinases/physiology , Haloperidol/pharmacology , Mitogen-Activated Protein Kinases/physiology , Prefrontal Cortex/physiology , Protein Phosphatase 2/physiology , Ribosomal Protein S6 Kinases, 90-kDa/physiology , Signal Transduction/drug effects , Animals , Blotting, Western , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/genetics , Immunoprecipitation , Male , Mitogen-Activated Protein Kinases/genetics , Phosphoprotein Phosphatases/metabolism , Phosphorylation/drug effects , Prefrontal Cortex/drug effects , Prefrontal Cortex/enzymology , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/physiology , Rats , Rats, Sprague-Dawley , Ribosomal Protein S6 Kinases, 90-kDa/genetics , raf Kinases/physiologyABSTRACT
Methamphetamine (MAP) is one of the most commonly abused drugs in Asia, and previous studies suggest that serotonin 3 receptors (5-HT(3)) are involved in MAP-induced locomotion and reward. However, little is known about the role of 5-HT(3) receptors in MAP-induced behavioral sensitization. Here, we measured the effects of MDL 72222, a 5-HT(3) antagonist, and SR 57227 A, a 5-HT(3) agonist, on the development and expression of MAP-induced behavioral sensitization, and alternations of 5-HT(3) receptor binding labeled with the 5-HT(3)-selective antagonist, [(3)H]GR65630, in mice. In addition, we investigated the effects of MAP on 5-HT(3A) receptor channel activity in Xenopus laevis oocytes expressing 5-HT(3A) receptors. We found that MDL 72222 attenuated both the development and expression of behavioral sensitization to MAP (1.0 mg/kg, i.p.), and that this attenuating effect of MDL 72222 was reversed by pre-treatment with SR 57227 A. In oocytes expressing 5-HT(3A) receptor, MAP exhibited a dual modulation of 5-HT(3A) receptor channel activity, i.e. pre-treatment with a low dose of MAP (0.1 microm) enhanced 5-HT-induced inward peak current (I(5-HT)) but a high dose of MAP (100 microm) inhibited I(5-HT). The acute administration of MDL 72222 with MAP decreased [(3)H]GR65630 binding versus MAP alone in the mouse striatum. Our results suggest that MDL 72222 attenuates MAP-induced behavioral sensitization via 5-HT(3) receptors in the caudate putamen, and that 5-HT(3) receptor antagonists like MDL 72222 have potential as novel anti-psychotic agents for the treatment of MAP dependence and psychosis.