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1.
Oncogene ; 33(4): 474-83, 2014 Jan 23.
Article in English | MEDLINE | ID: mdl-23318442

ABSTRACT

S6 kinases (S6Ks) are mechanistic target of rapamycin substrates that participate in cell growth control. S6Ks phosphorylate ribosomal protein S6 (rpS6) and additional proteins involved in the translational machinery, although the functional roles of these modifications remain elusive. Here we analyze the S6K-dependent transcriptional and translational regulation of gene expression by comparing whole-genome microarray of total and polysomal mouse liver RNA after feeding. We show that tissue lacking S6Ks 1 and 2 (S6K1 and S6K2), displays a defect in the ribosome biogenesis (RiBi) transcriptional program after feeding. Over 75% of RiBi factors are controlled by S6K, including Nop56, Nop14, Gar1, Rrp9, Rrp15, Rrp12 and Pwp2 nucleolar proteins. Importantly, the reduced activity of RiBi transcriptional promoters in S6K1;S6K2(-/-) cells is also observed in rpS6 knock-in mutants that cannot be phosphorylated. As ribosomal protein synthesis is not affected by these mutations, our data reveal a distinct and specific aspect of RiBi under the control of rpS6 kinase activity, that is, the RiBi transcriptional program.


Subject(s)
Protein Biosynthesis/physiology , Ribosomal Protein S6 Kinases/metabolism , Ribosomes/enzymology , Animals , Blotting, Northern , Blotting, Western , Gene Knock-In Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Phosphorylation , Real-Time Polymerase Chain Reaction , Ribosomal Protein S6/metabolism , Transcription, Genetic/physiology , Transcriptome
2.
Biogerontology ; 14(3): 303-23, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23686362

ABSTRACT

During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.


Subject(s)
Aging/physiology , Forkhead Transcription Factors/physiology , Insulin-Like Growth Factor I/physiology , Muscle, Skeletal/physiology , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/physiology , TOR Serine-Threonine Kinases/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Autophagy-Related Protein 7 , Female , Forkhead Box Protein O1 , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Models, Animal , Muscle Proteins/genetics , Muscle Proteins/physiology , SKP Cullin F-Box Protein Ligases/genetics , SKP Cullin F-Box Protein Ligases/physiology , Sarcopenia/physiopathology , Serpin E2/genetics , Serpin E2/physiology , Tripartite Motif Proteins , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/physiology , Young Adult
3.
Endocrinology ; 153(1): 241-53, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22087027

ABSTRACT

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS.


Subject(s)
Follistatin/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Receptor, IGF Type 1/metabolism , Animals , Base Sequence , DNA Primers/genetics , Follistatin/genetics , Humans , Hypertrophy , Insulin-Like Growth Factor II/deficiency , Insulin-Like Growth Factor II/genetics , Male , Mice , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Transfection
4.
Oncogene ; 29(7): 1003-16, 2010 Feb 18.
Article in English | MEDLINE | ID: mdl-19935711

ABSTRACT

The rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) is a key member of mTOR complex-2 (mTORC2), which phosphorylates the AGC kinases Akt/PKB, PKC and SGK1 at a C-terminal hydrophobic motif. We identified several novel sites on Rictor that are phosphorylated, including Thr1135, which is conserved across all vertebrates. Phosphorylation of this site on Rictor is stimulated by amino acids and growth factors through a rapamycin-sensitive signaling cascade. We demonstrate here that Rictor is a direct target of the ribosomal protein S6 kinase-1 (S6K1). Rictor phosphorylation at Thr1135 does not lead to major changes in mTORC2-kinase activity. However, phosphorylation of this site turns over rapidly and mediates 14-3-3 binding to Rictor and mTORC2, providing possibility for altered interactions of the complex. These findings reveal an unexpected signaling input into mTORC2, which is regulated by amino acids, growth factors and rapamycin.


Subject(s)
Carrier Proteins/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , 14-3-3 Proteins/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Cell Line , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Molecular Sequence Data , Phosphorylation , Rapamycin-Insensitive Companion of mTOR Protein , Rats , Threonine , Transcription Factors/metabolism
5.
Oncogene ; 29(11): 1641-52, 2010 Mar 18.
Article in English | MEDLINE | ID: mdl-19966861

ABSTRACT

Most cancer cells exhibit increased glycolysis for generation of their energy supply. This specificity could be used to preferentially kill these cells. In this study, we identified the signaling pathway initiated by glycolysis inhibition that results in sensitization to death receptor (DR)-induced apoptosis. We showed, in several human cancer cell lines (such as Jurkat, HeLa, U937), that glucose removal or the use of nonmetabolizable form of glucose (2-deoxyglucose) dramatically enhances apoptosis induced by Fas or by tumor necrosis factor-related apoptosis-inducing ligand. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. We established the fact that AMPK is activated upon glycolysis block resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease, but no other Bcl-2 anti-apoptotic members. Interestingly, we determined that, upon glycolysis inhibition, the AMPK-mTOR pathway controlled Mcl-1 levels neither through transcriptional nor through posttranslational mechanism but rather by controlling its translation. Therefore, our results show a novel mechanism for the sensitization to DR-induced apoptosis linking glucose metabolism to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of DR ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Apoptosis/physiology , Glycolysis/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Death Domain/metabolism , AMP-Activated Protein Kinases/antagonists & inhibitors , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Antibodies/immunology , Antibodies/pharmacology , Apoptosis/drug effects , Blotting, Western , Deoxyglucose/pharmacology , Enzyme Activation/drug effects , Glucose/pharmacology , Glycolysis/drug effects , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Models, Biological , Myeloid Cell Leukemia Sequence 1 Protein , Protein Biosynthesis/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA Interference , Receptors, Death Domain/immunology , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleotides/pharmacology , Sirolimus/pharmacology , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , TOR Serine-Threonine Kinases , U937 Cells , fas Receptor/immunology , fas Receptor/metabolism
6.
Nature ; 408(6815): 994-7, 2000.
Article in English | MEDLINE | ID: mdl-11140689

ABSTRACT

Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic beta cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4-8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in beta-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.


Subject(s)
Glucose Intolerance , Insulin/blood , Islets of Langerhans/ultrastructure , Ribosomal Protein S6 Kinases/metabolism , Animals , Blood Glucose/metabolism , Cell Size , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Fasting , Female , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Ribosomal Protein S6 Kinases/deficiency
7.
Croat Med J ; 40(3): 368-74, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10411964

ABSTRACT

The development of strategies which allow the inactivation of specific murine genes by homologous recombination in embryonic cells has revolutionized biological science in the last 10 years. A large number of mice carrying genetic lesions, generated by gene targeting technology, has tremendously increased our knowledge in many areas of biology, culminating in the identification of mouse models for human genetic disorders. These findings have been recently complemented by "conditional" gene targeting technology, allowing gene inactivation in a defined tissue and at a specific time point during development or adulthood, thereby extending the sophistication and potential of this technology.


Subject(s)
Gene Targeting , Genome , Animals , Disease Models, Animal , Gene Expression Regulation/genetics , Genetic Diseases, Inborn/genetics , Genetic Engineering , Genetic Vectors , Humans , Mice , Mice, Knockout/genetics , Recombination, Genetic
8.
EMBO J ; 17(22): 6649-59, 1998 Nov 16.
Article in English | MEDLINE | ID: mdl-9822608

ABSTRACT

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.


Subject(s)
Ribosomal Protein S6 Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary , Embryo, Mammalian/cytology , Embryonic and Fetal Development/genetics , Fibroblasts/cytology , Mice , Mice, Mutant Strains , Molecular Sequence Data , Phenotype , Phosphorylation , Ribosomal Protein S6 Kinases/chemistry , Ribosomal Protein S6 Kinases/metabolism , Sequence Homology, Amino Acid
9.
J Neurosci ; 17(4): 1291-301, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9006973

ABSTRACT

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Growth Substances/pharmacology , Neuroglia/metabolism , Neurotransmitter Agents/pharmacology , Stem Cells/metabolism , Animals , Calcium/metabolism , Calcium/physiology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carbachol/pharmacology , Enzyme Activation , Extracellular Space/physiology , Fibroblast Growth Factor 2/pharmacology , Ions , Kainic Acid/pharmacology , Phosphorylation , Protein Kinase C/physiology , Protein Serine-Threonine Kinases/metabolism , Rats/embryology , Ribosomal Protein S6 Kinases , Signal Transduction
10.
Eur J Neurosci ; 9(1): 93-101, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9042573

ABSTRACT

Kainic acid-induced seizures, in adult rats produce neurodegeneration in the hippocampus followed by sprouting of the mossy fibres in the inner molecular layer of the dentate gyrus and changes in GAP-43 expression in the granule cells. In the present study we observed that 4 days after kainic acid injection a dense plexus of silver-impregnated degenerating terminals detected by Gallyas's method and a decrease of GAP-43 immunostaining was observed in the inner molecular layer of the dentate gyrus indicating deafferentiation of this region. This was associated with the formation of an intense GAP-43 immunostained band in the supragranular layer. MK-801, a non-competitive inhibitor of the NMDA receptor, which partially inhibited the behavioural seizures induced by KA, also protected from the inner molecular layer deafferentation and markedly reduced the expression of GAP-43 mRNA in the granule cells and the intense GAP-43 immunostained band in the supragranular layer, suggesting a relationship among these events. Two months after kainic acid injection the intense supragranular GAP-43 positive band was no longer evident but the whole inner molecular layer appeared more labelled in association with the formation of the collateral sprouting of the mossy fibres in the inner molecular layer as detected by Timm's staining. These effects were also markedly reduced by the pretreatment with MK-801. Taken together, these experiments indicate for the first time a direct relationship between the increase of GAP-43 immunostaining in the inner molecular layer of the dentate gyrus and the collateral sprouting of mossy fibres in this district in response to kainic acid induced seizures. This further supports the hypothesis that the early induction of GAP-43 in granule cells may be one of the molecular mechanisms required for the synaptic reorganization of the mossy fibres.


Subject(s)
Dentate Gyrus/metabolism , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/physiology , Kainic Acid/pharmacology , Membrane Glycoproteins/biosynthesis , Nerve Fibers/physiology , Nerve Tissue Proteins/biosynthesis , Neurofilament Proteins/biosynthesis , Synapses/physiology , Animals , Dentate Gyrus/drug effects , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , GAP-43 Protein , Hippocampus/cytology , Hippocampus/drug effects , Immunohistochemistry , In Situ Hybridization , Male , Nerve Fibers/drug effects , Pyramidal Cells/physiology , Rats , Rats, Sprague-Dawley , Seizures/chemically induced , Seizures/pathology , Silver Staining , Synapses/drug effects
11.
Neuroreport ; 7(15-17): 2539-42, 1996 Nov 04.
Article in English | MEDLINE | ID: mdl-8981419

ABSTRACT

We have previously shown that kainic acid-induced seizures in adult rats caused an up-regulation of GAP-43 mRNA in the granule cells of the hippocampus, suggesting an involvement of this protein in the kainic acid-induced sprouting of mossy fibres. To determine whether this effect was dependent on the synthesis of proteins activated under these experimental conditions we examined the effect of cycloheximide, a protein synthesis inhibitor, on kainic acid-induced GAP-43 mRNA. Cycloheximide, injected s.c. 2 h but not 8 h after kainic acid, markedly reduced the increased expression of GAP-43 mRNA in granule cells. These results suggest that a rapid mechanism involving new protein synthesis is activated by kainic acid to induce GAP-43 in the granule cells and possibly trigger the structural remodeling of mossy fibres.


Subject(s)
Cycloheximide/pharmacology , Dentate Gyrus/drug effects , Kainic Acid/pharmacology , Membrane Glycoproteins/drug effects , Nerve Tissue Proteins/drug effects , Animals , Autoradiography , Dentate Gyrus/metabolism , GAP-43 Protein , Male , Rats , Rats, Sprague-Dawley
12.
Neurochem Res ; 20(5): 549-60, 1995 May.
Article in English | MEDLINE | ID: mdl-7643960

ABSTRACT

Here we review experimental evidence of non-NMDA glutamate receptor expression in the embryonic central nervous system. AMPA- and kainate-preferring glutamate receptor subunit mRNA transcripts are detected in embryonic neurons, glia and neural progenitors. Functional assays demonstrate that in some cell subpopulations ionotropic glutamate receptors are expressed by progenitors before synapse formation and terminal differentiation, and may be present before lineage determination is specified. The activation of these receptors triggers induction of immediate early gene transcription in progenitor cells. The cloning and transcriptional analysis of upstream regulatory regions of glutamate receptor genes governing their temporal and tissue-specific expression are also discussed.


Subject(s)
Neuroglia/metabolism , Neurons/metabolism , Receptors, AMPA/biosynthesis , Receptors, Kainic Acid/biosynthesis , Stem Cells/metabolism , Animals , Cells, Cultured , Humans , Neuroglia/cytology , Neurons/cytology , Receptors, Glutamate/biosynthesis , Tumor Cells, Cultured
13.
Eur J Neurosci ; 6(4): 509-15, 1994 Apr 01.
Article in English | MEDLINE | ID: mdl-8025706

ABSTRACT

The axonal growth-associated protein GAP-43 is believed to play some role in the synaptic remodelling that takes place in the hippocampus of adult rats after certain experimental lesions. GAP-43 mRNA is highly expressed in adult CA3 pyramidal cells but almost absent in the dentate granule cells. We analysed whether the sprouting of granule cell axons, the mossy fibres of the hippocampus, caused by kainic acid-induced seizures in adult rats was associated with any induction of GAP-43 mRNA in granule cells and with any changes in the immunostaining pattern of GAP-43 in the hippocampus. Increased GAP-43 mRNA expression was found to be induced in granule cells 18, 24 and 30 h after a systemic injection of kainic acid which induced generalized seizures in adult rats, and returned to control levels by 48 h post-treatment. No effect was observed in other regions of the hippocampus. However, when kainic acid was injected into 15-day-old rats, which responded with generalized seizures but no sprouting of mossy fibres, there was no induction of GAP-43 mRNA in the granule cells, suggesting a close relation between GAP-43 expression and sprouting of these cells. Seven days after kainic acid injections, GAP-43 immunostaining was decreased in the inner molecular layer of the dentate gyrus except for a thin supragranular band, whereas 30 days after treatment all animals showed increased GAP-43 immunoreactivity in the whole inner molecular layer.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Hippocampus/metabolism , Membrane Glycoproteins/biosynthesis , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Pyramidal Cells/metabolism , Seizures/metabolism , Animals , GAP-43 Protein , Hippocampus/cytology , Hippocampus/pathology , Immunohistochemistry , In Situ Hybridization , Kainic Acid , Male , Membrane Glycoproteins/analysis , Nerve Fibers/pathology , Nerve Fibers/physiology , Nerve Tissue Proteins/analysis , Neurofilament Proteins/biosynthesis , Neurons/cytology , Neurons/pathology , Pyramidal Cells/cytology , Pyramidal Cells/pathology , Rats , Rats, Sprague-Dawley , Reference Values , Seizures/chemically induced , Seizures/pathology , Time Factors
14.
Proc Natl Acad Sci U S A ; 91(8): 3215-9, 1994 Apr 12.
Article in English | MEDLINE | ID: mdl-8159727

ABSTRACT

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.


Subject(s)
Calcium/metabolism , DNA-Binding Proteins/genetics , Genes, Immediate-Early , Glutamates/pharmacology , Immediate-Early Proteins , Oligodendroglia/cytology , Receptors, AMPA/physiology , Transcription Factors/genetics , Animals , Early Growth Response Protein 1 , Gene Expression/drug effects , Genes, fos , Genes, jun , Potassium/physiology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, Kainic Acid/physiology
15.
Brain Res Bull ; 34(4): 389-94, 1994.
Article in English | MEDLINE | ID: mdl-8082031

ABSTRACT

Intracerebroventricularly (ICV) injected 5,7-dihydroxytryptamine (5,7-DHT), which reduced by 70-90% forebrain serotonin levels, significantly raised glial fibrillary acidic protein (GFAP) mRNA levels in the hippocampus and nucleus raphe dorsalis 5 days but not 15 days after the lesion. A significant increase of mitochondrial benzodiazepine receptors (MBR), measured by binding autoradiography of 3H-PK 11195, was found in the nucleus raphe dorsalis 5 and 15 days after the ICV 5,7-DHT and also in the hippocampus, ventral tegmental area, and substantia nigra at 15 days. No significant effect was observed in the striatum and cortex for either GFAP mRNA or MBR binding. Unlike the ICV route, bilateral injection of 5,7-DHT into the medial forebrain bundle, which caused a 65-90% reduction of serotonin levels in different forebrain regions, significantly raised GFAP mRNA and MBR binding only at the site of injection with no effect in hippocampus, striatum, and cortex. MBR binding slightly increased in the nucleus raphe dorsalis 15 days after the lesion. High doses of d-fenfluramine (10 mg/kg intraperitoneally twice daily for 4 days) caused 80-90% reduction of serotonin levels 5 days after the last injection but did not change the GFAP mRNA or the MBR binding in any of the brain regions considered. These findings suggest that the effect of 5,7-DHT on microglial and glial markers is probably related to a nonspecific interaction with other neuronal systems besides the serotonin or to direct interaction with glial cells; the use of these parameters for detecting selective degeneration of serotonin axons presents some obvious limitations.


Subject(s)
Glial Fibrillary Acidic Protein/biosynthesis , Mitochondria/metabolism , Nerve Degeneration/physiology , Neurons/metabolism , Neurons/physiology , RNA, Messenger/biosynthesis , Receptors, GABA-A/metabolism , Serotonin/physiology , 5,7-Dihydroxytryptamine/administration & dosage , 5,7-Dihydroxytryptamine/pharmacology , Animals , Autoradiography , Blotting, Northern , Brain/anatomy & histology , Brain Chemistry/drug effects , In Situ Hybridization , Injections , Injections, Intraventricular , Isoquinolines , Male , Medial Forebrain Bundle , Mitochondria/drug effects , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/drug effects , Serotonin/metabolism
16.
Brain Res ; 604(1-2): 325-30, 1993 Feb 26.
Article in English | MEDLINE | ID: mdl-8096158

ABSTRACT

The depolarization-evoked release of endogenous glutamate (GLU) and -aspartate (ASP) and its modulation mediated by gamma-aminobutyric acid (GABA) heteroreceptors was investigated in superfused rat cerebrocortical synaptosomes. Exposure to 12 mM K+ enhanced the release of GLU and ASP. The K(+)-evoked overflow of both amino acids was largely Ca(2+)-dependent. Exogenous GABA inhibited the K(+)-evoked overflow of GLU (EC50 2.8 microM) and ASP (EC50 2.7 microM). The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (EC50 2.0 microM for GLU and 1.3 microM for ASP release) but not by the GABAA receptor agonist muscimol, up to 100 microM. Accordingly, the GABA-induced inhibition of GLU and ASP release was not affected by the GABAA receptor antagonists, bicuculline or picrotoxin, but was antagonized by the GABAB receptor antagonist, 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP 35348). The GABA effect was, however, insensitive to another GABAB receptor antagonist, phaclofen, up to 1,000 microM. It can be concluded that GABA heteroreceptors of the GABAB type regulating the depolarization-evoked release of GLU and ASP are present on cortical GLU/ASP-releasing nerve terminals. These receptors may be classified as a phaclofen-insensitive GABAB receptor subtype.


Subject(s)
Aspartic Acid/metabolism , Cerebral Cortex/physiology , Glutamates/metabolism , Receptors, GABA-A/physiology , Synaptosomes/metabolism , gamma-Aminobutyric Acid/pharmacology , Animals , Baclofen/analogs & derivatives , Baclofen/pharmacology , Bicuculline/pharmacology , GABA-A Receptor Antagonists , Glutamic Acid , Kinetics , Male , Organophosphorus Compounds/pharmacology , Picrotoxin/pharmacology , Potassium Chloride/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/classification , Synaptosomes/drug effects
17.
Br J Pharmacol ; 107(4): 1146-51, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1361400

ABSTRACT

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices. The effect of clonidine (0.01- 1 g1M) on the evoked overflow of tritium was reduced in presence of 1 muM desipramine.9. It is proposed that autoregulation of NA release can occur in human cerebral cortex. The process involves activation of alpha 2-adrenoceptors which may be either the alpha2X or the alpha2D subtype.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Cerebral Cortex/metabolism , Norepinephrine/metabolism , Receptors, Adrenergic, alpha/classification , Adult , Aged , Calcium/metabolism , Cerebral Cortex/drug effects , Clonidine/pharmacology , Desipramine/pharmacology , Dose-Response Relationship, Drug , Electric Stimulation , Female , Humans , In Vitro Techniques , Male , Middle Aged , Oxymetazoline/pharmacology , Receptors, Adrenergic, alpha/metabolism , Tetrodotoxin/pharmacology
18.
J Neurochem ; 59(4): 1481-9, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1402899

ABSTRACT

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Axons/metabolism , Carrier Proteins/metabolism , Central Nervous System/metabolism , Glycine/physiology , gamma-Aminobutyric Acid/physiology , Animals , Cerebellum/metabolism , Cerebral Cortex/metabolism , Glycine/metabolism , Hippocampus/metabolism , Male , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , gamma-Aminobutyric Acid/metabolism
19.
Neurosci Lett ; 136(1): 127-30, 1992 Feb 17.
Article in English | MEDLINE | ID: mdl-1321965

ABSTRACT

The electrically-evoked release of [3H]dopamine ([3H]DA) from rat striatal slices was studied after a monolateral intrastriatal injection of kainic acid (KA). The release in the KA-lesioned striatum measured 4 days after the lesion was largely reduced (by 80%) with respect to the contralateral striatum. Administration of GM1 ganglioside (GM1) beginning on the day of the lesion resulted in restoration of the catecholamine release. Significant recovery was observed when GM1 was administered i.p. daily at the dose of 3 mg/kg for 6 days. The ganglioside given for 6 days at 30 mg/kg restored to near normal the electrically-evoked [3H]DA release. Similar recovery from the KA-induced injury occurred spontaneously but required 50 days.


Subject(s)
Corpus Striatum/drug effects , Dopamine/metabolism , G(M1) Ganglioside/pharmacology , Kainic Acid/toxicity , Animals , Corpus Striatum/metabolism , Denervation , Electric Stimulation , Male , Neurons/metabolism , Rats , Rats, Inbred Strains , Receptors, Kainic Acid , Receptors, Neurotransmitter/physiology
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