Takahiro Ozaki

Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3.

The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin.

Distribution of Fos- and Jun-related proteins and activator protein-1 composite factors in mouse brain induced by neuroleptics

The mechanisms by which the direct actions of neuroleptics are translated into therapeutic effects are unknown. We immunocytochemically investigated the expression of Fos- and Jun-related proteins and examined activator protein-1 DNA-binding activity in ddY mouse brain 120 min after the administration of haloperidol (1 mg/kg), (-)-sulpiride (20 mg/kg) and a selective dopamine D1 receptor antagonist, SCH23390 (1 mg/kg). The densities of Fos-, FosB-, Fra-1-, Jun- and JunD-immunoreactive nuclei induced by haloperidol and sulpiride in the hippocampus, piriform cortex and accumbens nucleus were higher than those in the control groups. The same regions showed higher densities of FosB-, Fra-1- and JunD-immunoreactive nuclei induced by SCH23390 compared with the control groups. We investigated further the activator protein-1 composite factors using super gel shift assays. These results suggested that induced Fos, FosB, Fra-1, Jun and JunD proteins constitute the activator protein-1 complex after the administration of haloperidol and sulpiride. In contrast, FosB, Fra-1 and JunD appear to constitute the activator protein-1 complex after the administration of SCH23390. Therefore, the diversity of activator protein-1 composite factors suggests that various kinds of gene are induced to act by some neuroleptics.

Comparison of the effects of dopamine D1 and D2 receptor antagonists on nerve growth factor mRNA expression

Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated by the interaction of c-fos with an activator protein-1 (AP-1) binding site present in the first intron on the NGF gene. Using an RNase protection assay and in situ hybridization, we examined the effects of dopamine D1 and D2 receptor antagonists on NGF mRNA. Haloperidol (0.1-8 mg/kg) and (-)-sulpiride (10-100 mg/kg), induced NGF mRNA in a dose-dependent fashion in the hippocampus, piriform cortex, striatum and nucleus accumbens. The haloperidol (1 mg/kg)- and (-)-sulpiride (20 mg/kg)-induced NGF mRNA expression attained a maximum level 120 min after injection and returned to control levels 24 h later. Prior administration of the protein synthesis inhibitor cycloheximide blocked the haloperidol- and (-)-sulpiride-mediated induction of NGF mRNA. In contrast, R-(-)-8-chloro-2,3,4,5-tetrahydro-3,1-methyl-5-phenyl-11-3-benzyoepin e-7-ol (SCH23390) did not induce NGF mRNA expression in either a dose-dependent or time-dependent manner. Our previous studies have shown that haloperidol and (-)-sulpiride induce the expression of c-fos and c-jun mRNAs and increase their AP-1 DNA binding activities. Thus, the data suggest that neuroleptics induce NGF gene expression by increasing AP-1 DNA binding activity.

Comparative effects of dopamine D1 and D2 receptor antagonists on nerve growth factor protein induction

We previously reported that following acute administration of haloperidol or (-)-sulpiride, both dopamine D(2)-receptor antagonists, to mice induced nerve growth factor (NGF) gene expression, mediated by the interaction of c-fos with the AP-1 binding site present in the first intron on the NGF gene. In contrast, the D(1)-receptor antagonist R-(-)-8-chloro-2,3,4, 5-tetrahydro-3,1-methyl-5-phenyl-11-3-benzyoepine-7-ol (SCH23390) did not induce NGF mRNA expression. We report here immunohistochemical and Western blot analyses showing that following injection of these drugs for 14 consecutive days, the amount of NGF protein increased gradually and was induced significantly in the hippocampus, piriform cortex, amygdala, dorsal striatum, and nucleus accumbens neurons. NGF enhances the release of acetylcholine from these regions. Cholinergic innervation in the striatum and nucleus accumbens neurons is believed to be related to late-onset extrapyramidal symptoms, while in the hippocampus and piriform cortex it is involved in enhancing cognition. Thus, our data suggest that haloperidol- and (-)-sulpiride-induced NGF expression may be associated with both beneficial and adverse effects.

The comparative effects of haloperidol, (−)-sulpiride, and SCH23390 on c-fos and c-jun mRNA expressions, and AP-1 DNA binding activity

The c-fos and c-jun mRNA expressions were measured by the RNase protection assay method following intraperitoneal injection of haloperidol, (D1 and D2 receptor antagonists), (-)-sulpiride, (D2 receptor antagonist), and SCH23390, (D1 receptor antagonist). Haloperidol and (-)-sulpiride increased their mRNA expressions in a dose-dependent manner, peaking at 30 min after injection followed by a gradual decline. The SCH23390 did not induce expression of either c-fos or c-jun mRNA. A significant decrease of c-fos as well as c-jun mRNA expression was found due to pretreatment with SCH23390 (1 mg/kg i.p.) followed by injection of (-)-sulpiride (20 mg/kg i.p.). The results suggest that the expression of these mRNAs is closely related to the dopamine D2-like antagonism. Administration of haloperidol or (-)-sulpiride increased AP-1 DNA binding activity with similar manner of dose-dependence, whereas their activities were reduced by Fos and Jun antibodies, implying that AP-1 components, transcriptional factors, forming due to Fos and Jun were actually activated by either haloperidol or (-)-sulpiride.

Alterations of Glutamate Transporter Gene Expression in Epileptic EL Mice Brains

Intmduc%ion: Glutamate is considered to be an important excitatory amino acid and has been implicated in the pathophysiology of cpilcpsy. Glutamate transporters help to maintain the extracellular glutamate concentration and have been identified i n inousc brains, incl tiding GLT‐I and GLAST in astroglia and EAAC‐I in neurons. In an amygdala‐kindling study, alteration of glutamate transporter proteins showed that GLAST protein was down‐regulated in the piriform cortcx/amygclala rcgion of the amygdala‐kindled Iiits. In contrast, kindling induced an increase in EAAC‐ I levels in the pirilorrn cortex/amygdala and hippocampus. No changes i n GLT‐I wcrc obscrvcd in any region. The EL mouse, an inbred mutant strain, is considered to be a model for human complex partial seidures. This study was carried out to reveal the relationship bctwccn cpilcptogcncsis and glutamate transporters in EL mice.

Effects of a Pentylenetetrazol‐Induced Convulsion on Mouse Cortical Protein Kinase C and Ca2+/Calmodulin‐Dependenl Protein Kinase I1 Activity.

and CBZ, may possibly enhance the transformations of AEDs into epoxide, which may result in increased AED teratogenesis. On the other hand, the effects of AEDs on the detoxification or elimination process of epoxide have not yet been fully clarified. Our study clearly showed that therapeutic concentrations of VPA show inhibitory effects on the activities of EH and most of the GST isozymes, and that these effects are concentration dependent. Therapeutic concentrations of CBZ also inhibited the activity of GST 7-7, found primarly in the placenta. These results suggest that detoxification of the epoxide of AEDs could be inhibited under AED polytherapy by using VPA or CBZ or both. This may partly explain the increased incidence of malformations in offspring exposed to VPA-CBZ, administered with or without other AEDs. PHT, PB, and ZNS had no inhibitory effects on the activities of EH and GST. However, the possibility that PHT and PB have inducing effects on epoxide formation should be taken into account. In this regard, combinations of AEDs should be carefully selected in the treatment of women of childbearing age with epilepsy. (This work was supported by Grant-in-aid No. 05454309 for Scientific Research from the Ministry of Education and Culture and grant from the Hirosaki Research Institute for Neurosciences.)

Alteration of Fos-and Jun-Related Antigens After Seizures in Epileptic EL Mice.

analysis with anti-PKC-isozyme-specific antibodies was studied. The content of 80-K PKC /3 in the cytosolic fraction increased, whereas that in the membrane fractions decreased at 10 min after convulsion. The SO-K PKC /3 was detected in the cytosolic fraction during the tonic convulsion and 10 min after the convulsion phase. The findings in immunoblot analysis obtained by using anti-PKC a specific antibodies were similar to those obtained by using anti-PKC /3 specific antibodies, but those of PKC y with polyclonal proteins were not altered by the PTZ-induced convulsion. Our findings suggest that cytosolic PKC activity is increased by translocation of PKC a and p from the membrane to cytosol and release of 50-K PKC fragments. Although we could not demonstrate the mechanism of activation of membranous CaMKII by a PTZ convulsion, the activation of PKC in the cytosol and CaMKII activity in the membrane may be involved in the mechanism of the convulsion, such as termination of the convulsion or intracellular changes such as expression of immediate early genes.

The alteration of AP-1 DNA-binding activity in seizure-prone EL mouse brain.

The EL mouse being an inbred mutant strain is thought to be a model for human complex partial seizures.’V2 Our previous studies have shown that c

P-4-6 The effect of neuroleptics on the AP-1 DNA binding activity and the induction of fos and jun

os mRNA and Fos expressions in EL mouse brain by toss-up induced seizures prolonged more than those in EL and ddY mice by bemegride-induced ones.3 The results suggest that extended c