Yasutaka Azuma

Particular nuclear transcription factors responsive to systemic administration of kainic acid in murine brain.

Gel retardation electrophoresis revealed that binding of a radiolabeled double stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 (AP1) was markedly potentiated 2 h after the intraperitoneal injection of kainic acid (KA) at a dose range of 10-40 mg/kg in a dose-dependent manner in the murine hippocampus. The potentiation was seen in a manner independent of the crisis of convulsive seizures following the administration of KA at different doses. At the highest dose employed, the systemic KA significantly potentiated the AP1 binding in most central discrete structures examined except the cerebellum. In contrast, KA significantly potentiated binding of a radiolabeled probe for cyclic AMP response element binding protein (CREB) in a dose-dependent fashion in the hippocampus, without altering that in other parts of murine brain. No significant alteration was detected in binding of a probe for c-Myc in any brain regions examined 2 h after the administration of KA at different doses. However, immunoblotting analysis demonstrated that KA was ineffective in altering endogenous levels of both CREB and CREB phosphorylated at serine133 in the hippocampus and cerebellum. These results suggest that in vivo systemic KA signals may be selectively transduced to nuclear AP1 in the hippocampus through a mechanism different from phosphorylation of CREB at serine133 in murine brain.

Predominant expression of nuclear activator protein-1 complex with DNA binding activity following systemic administration of N-methyl-d-aspartate in dentate granule cells of murine hippocampus

The systemic administration of N-methyl-D-aspartate (100 mg/kg, i.p.) resulted in preferential but transient expression of the transcription factor activator protein-1 in the granule cell layers of the dentate gyrus in the murine hippocampus by maximally 700% 1 h later, without markedly affecting that in the pyramidal cell layers of the CA1 and CA3 subfields for 4 h. The potentiation was completely prevented by prior administration of the N-methyl-D-aspartate channel blocker dizocilpine at 10 mglkg. By contrast, kainate (40 mg/kg, i.p.) potentiated activator protein-1 DNA binding in adjacent areas around the pyramidal and granule cell layers, in addition to potentiating that in neuronal cell layers of the CA1 and CA3 subfields and the dentate gyrus. Light microscopic analysis revealed that kainate, but not N-methyl-D-aspartate, induced marked losses of the pyramidal cells in the CAI and CA3 subfields, without affecting the dentate granule cells, for 14 days after administration. Limited proteolysis by V8 protease and supershift, as well as immunoblotting assays using antibodies against c-Fos and c-Jun, invariably gave support for differential expression by N-methyl-D-aspartate and kainate of the activator protein-1 complex consisting of different partner proteins. Moreover, two-dimensional electrophoresis followed by immunoblotting analysis revealed the expression of several nuclear proteins immunoreactive with the anti-c-Fos antibody at molecular weights and isoelectric points clearly different from those of c-Fos itself in response to kainate, but not N-methyl-D-aspartate, in the hippocampus. These results suggest that in vivo N-methyl-D-aspartate signals are predominantly transduced into cell nuclei to express activator protein-1 complex through molecular mechanisms different from those for kainate signals in the granule cells of the dentate gyrus in the murine hippocampus.

Positive correlation between prolonged potentiation of binding of double-stranded oligonucleotide probe for the transcription factor AP1 and resistance to transient forebrain ischemia in gerbil hippocampus

Gel retardation electrophoresis revealed that binding of a radiolabelled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 was markedly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils with transient forebrain ischemia for 5 min, which is known to induce delayed death of pyramidal neurons exclusively in the CA1 subfield. The potentiation was transient in the vulnerable CA1 subfield, but persistent up to 18 h in the resistant CA3 subfield and dentate gyrus. However, no significant alteration was detected in endogenous levels of cyclic AMP response element binding protein phosphorylated at serine133 in these three different hippocampal structures 3 h after the reperfusion. On the other hand, hypothermia during ischemia which is known to protect the CA1 subfield against ischemic damages, led to a prolonged elevation of the activator protein-1 binding up to 9 h after the reperfusion in this vulnerable subfield at least in part through expression of c-Fos protein. Moreover, activator protein-1 binding was significantly elevated in the CA1 subfield up to 12 h after forebrain ischemia for 2 min which is shown not to induce marked damages to the vulnerable subfield. These results suggest that prolonged elevation of DNA binding activity of activator protein-1 may be responsible for molecular mechanisms underlying the unique vulnerability and/or resistance of particular subfields to a transient ischemic insult in the gerbil hippocampus.

Constitutive expression of cytoplasmic activator protein-1 with DNA binding activity and responsiveness to ionotropic glutamate signals in the murine hippocampus.

Gel retardation electrophoresis revealed that cytosolic fractions contained DNA binding activity of the transcription factor activator protein-1 with profiles different from those reported in nuclear extracts in murine brain. In particular, activator protein-1 DNA binding was almost undetectable at 25 degrees C in the presence of both KCl and MgCl2 in cytosol fractions. Moreover, cytoplasmic activator protein-1 binding occurred at three different mobilities on the gel when determined at 2 degrees C in the absence of MgCl2. Systemic administration of N-methyl-D-aspartate and kainate led to marked potentiation of cytoplasmic activator protein-1 binding detected as slow bands in the murine hippocampus, without markedly affecting that as a fast band. Immunoblotting and supershift assays revealed much higher expression of both immunoreactive c-Jun and c-Fos in hippocampal cytosolic fractions in response to the administration of kainate than N-methyl-D-aspartate. These results suggest that activator protein-1 may be constitutively expressed in the cytoplasm with DNA binding activity and responsiveness to ionotropic glutamate signals in a manner different from that in the nucleus in the murine hippocampus.

N-methyl-d-aspartate signaling to nuclear activator protein-1 through mechanisms different from those for kainate acid signaling in murine brain

Protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in cell nuclei in eukaryotes. The systemic administration of N-methyl-D-aspartate resulted in selective but transient potentiation of binding of a radiolabeled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 in murine hippocampus, without markedly affecting binding of probes for other transcription factors. By contrast, kainic acid induced more potent and more persistent potentiation of activator protein- binding in the hippocampus than N-methyl-D-aspartate. The protein synthesis inhibitor cycloheximide was effective in significantly preventing the potentiation by N-methyl-D-aspartate, but not that by kainic acid at the doses used. Moreover, kainic acid induced much more and longer expression of immunoreactive c-Fos protein in the hippocampus than N-methyl-D-aspartate. However, neither N-methyl-D-aspartate nor kainate induced expression of cyclic AMP response element binding protein phosphorylated at serine133 in the hippocampus from 10 min to 24 h after the administration. Instead, kainate was more potent than N-methyl-D-aspartate in facilitating both dephosphorylation at serine and phosphorylation at tyrosine of particular nuclear proteins in the hippocampus. These results suggest that N-methyl-D-aspartate and kainate signals may be differentially transduced into cell nuclei to express the activator protein-1 complex through molecular mechanisms which differ from phosphorylation of cyclic AMP response element binding protein at serine133 but involve serine dephosphorylation and/or tyrosine phosphorylation of particular nuclear proteins in the murine hippocampus.

Possible involvement of activator protein-1 DNA binding in mechanisms underlying ischemic tolerance in the CA1 subfield of gerbil hippocampus

Transcription factors are nuclear proteins with an ability to recognize particular nucleotide sequences on double stranded genomic DNAs and thereby modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNAs in cell nuclei. Gel retardation electrophoresis revealed that transient forebrain ischemia for 5 min led to drastic potentiation of binding of a radiolabelled double-stranded oligonucleotide probe for the transcription factor activator protein-1, in the thalamus as well as the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils previously given ischemia for 2 min two days before, which is known to induce tolerance to subsequent severe ischemia in the CA1 subfield. By contrast, ischemia for 5 min resulted in prolonged potentiation of activator protein-1 binding in the vulnerable CA1 subfield of the gerbils with prior ischemia for 5 min 14 days before, which is shown to induce delayed death of the pyramidal neurons exclusively in this subfield. Similar prolongation was seen with activator protein-1 binding in the vulnerable thalamus but not in the resistant CA3 subfield and dentate gyrus of the gerbils with such repeated ischemia for 5 min. Limited proteolysis by Staphylococcus aureus V8 protease as well as supershift assays using antibodies against c-Fos and c-Jun proteins demonstrated the possible difference in constructive partner proteins of activator protein-1 among nuclear extracts of the CA1 subfield obtained from gerbils with single, tolerated and repeated ischemia. These results suggest that de novo protein synthesis may underlie molecular mechanisms associated with acquisition of the ischemic tolerance through modulation at the level of gene transcription by activator protein-1 composed of different constructive partner proteins in the CA1 subfield. Possible participation of glial cells in the modulation is also suggested in particular situations.

Differentiation by magnesium ions of affinities of nuclear proteins for consensus core nucleotide element of the transcription factor c-Myc in murine brain

The addition of divalent cations such as Mg2+ and Ca2+ ions markedly reduced binding of a radiolabeled double stranded oligonucleotide probe for the transcription factor c-Myc in the presence of 100 mM KCl in nuclear extracts of the mouse whole brain. Irrespective of the addition of MgCl2, binding was selectively competed with the unlabeled probe for c-Myc having a double stranded conformation. Treatment with V8 protease differentially modulated binding of the probe for c-Myc determined in the presence and absence of added MgCl2. Introduction of irreversible covalent bonding between the radiolabeled probe and nuclear proteins led to retarded mobility of the radioactive probe/protein complex in the presence of MgCl2 on sodium dodecyl sulfate electrophoresis regardless of treatment with DNase. However, an antibody against the c-Myc protein affected neither mobility nor intensity of the radioactive band on gel retardation electrophoresis. Moreover, regional distribution was different from each other in mouse brain when determined in the presence and absence of added MgCl2. These results suggest that magnesium ions may have an ability to differentiate between nuclear c-Myc family proteins with different affinities for the consensus core nucleotide element CACGTG in murine brain.

Binding of double stranded oligonucleotide probes for particular transcription factors with leucine-zipper motifs in discrete brain structures of mice with acquired and inherent spontaneous seizures

Nuclear extracts of mouse brain contained binding of radiolabeled oligonucleotide probes for particular transcription factors with leucine-zipper motifs including activator protein-1 (AP1), cyclic AMP response element binding protein (CREB) and c-Myc. An acute intraperitoneal injection of pentylenetetrazole (PTZ) at a convulsive dose significantly potentiated binding of the probe for AP1 in the cerebral cortex, hippocampus, striatum, hypothalamus and midbrain, without affecting that in the medulla-pons and cerebellum, 2 h after the administration. However, PTZ failed to affect binding of the probe for CREB under the similar experimental conditions. In contrast, PTZ induced a slight but statistically significant decrease in binding of the AP1 probe in the cerebellum, without altering that in the hippocampus, 14 h after the injection. On the other hand, repeated administration of PTZ at a subconvulsive dose led to spontaneous kindling seizures in animals, with a concomitant decrease in binding of the AP1 probe in both the hippocampus and cerebellum. In contrast to these animals with acquired spontaneous seizures, however, binding of the AP1 probe was significantly higher in three different telencephalic structures of inherently spontaneous epileptic El mice than that in the parent ddY mice, with binding of probes for CREB and c-Myc being unchanged. These results suggest that different molecular mechanisms may underlie the expression of being unchanged. These results suggest that different molecular mechanisms may underlie the expression of AP1 in discrete brain structures of mice with acquired and inherent spontaneous seizures.

Correlation between potentiation of AP1 DNA binding and expression of c-Fos in association with phosphorylation of CREB at serine133 in thalamus of gerbils with ischemia.

Protein biosynthesis is mainly under the control at the level of gene transcription in eukaryotes. Transcription factors are nuclear proteins with abilities to modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNA from double stranded DNA in the cell nuclei. Binding of a radiolabeled oligonucleotide probe for the transcription factor activator protein-1 (AP1) was transiently potentiated 1 to 6 h after the recirculation of blood supply in the thalamus and striatum, but not in the entorhinal cortex, olfactory bulb, frontal cortex, cerebellar cortex and medulla-pons, in gerbils with transient global forebrain ischemia for 5 min, in addition to the hippocampal subregions. The ischemic insult not only increased the immunoreactivity with an antibody against cyclic AMP response element binding protein (CREB) phosphorylated at serine133, but also induced the expression of both c-Jun and c-Fos family proteins 3 h after the recirculation in the thalamus. Limited proteolysis by Staphylococcus aureus (S. aureus) V8 protease revealed the expression of different partner proteins of AP1 in response to ischemic signals in the thalamus. Moreover, ischemia for 2 min led to more prolonged elevation of AP1 binding in the thalamus at least up to 12 h after the reperfusion than that seen with ischemia for 5 min. These results suggest that potentiation of AP1 DNA binding may at least in part involve mechanisms associated with the expression of c-Fos protein through phosphorylation of CREB at serine133 in the thalamus of gerbils with ischemia.

Discrimination by added ions of ligands at ionotropic excitatory amino acid receptors insensitive to N-methyl-D-aspartate in rat brain using membrane binding techniques.

The addition of potassium thiocyanate almost quadrupled binding of [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to an AMPA-sensitive subclass of brain excitatory amino acid receptors in rat brain synaptic membranes, treated with Triton X-100. Among several ligands tested, quisqualic acid (QA) was the most potent displacer of [3H]AMPA binding in the absence of added SCN- ions, followed by AMPA, 6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), glutamic (Glu) and kainic (KA) acids in a rank order of decreasing potency. The addition of SCN- ions was effective in significantly reducing the potencies of antagonists such as DNQX and CNQX, without affecting those of agonists including QA, AMPA, Glu and KA. On the other, the addition of Ca2+ ions significantly inhibited [3H]KA binding in a concentration-dependent manner at concentrations of above 2.5 mM. Calcium ions were also effective in significantly potentiating potencies to displace [3H]KA binding of antagonists such as DNQX and CNQX, with concomitant reduction of those of agonists including KA, QA and Glu. However, N-methyl-D-aspartic acid (NMDA) did not affect binding of both radioligands at concentrations of below 0.1 mM. These results suggest that both SCN- and Ca2+ ions may be useful to discriminate agonists and antagonists among a variety of displacers of ligand binding to the non-NMDA receptors in the brain.