Using a DNA microarray method to examine gene expression in brain from clozapine-injected mice.

After our 2002 report on the changes of gene expression in the brain from phencyclidine-treated mouse by using DNA microarray (Toyooka et al., Ann. N.Y. Acad. Sci. 965: 10-20), we decided to apply this DNA microarray method for the brain of the mouse treated with other drugs. We are now examining the effects of clozapine on the brain function. Clozapine is an atypical antipsychotic drug. Clozapine has affinity for neurotransmitter receptors, including D4 dopamine, serotonin, histamine, and adrenergic receptors. MacGibbon et al. (Mol. Brain Res. 23: 21-32) reported that clozapine and haloperidol produced a differential pattern of immediate early gene expression in rat caudate-putamen. Kobayashi et al. (Br. J. Pharmacol. 123: 421-426) have observed the effects of clozapine on the opioid receptors and G-protein-activated inwardly rectifying potassium channel of Xenopus oocytes. Thomas et al. (J. Neurochem. 76: 789-796) found that clozapine increases apolipoprotein D expression in the mouse brain. Therefore, we are going to apply this DNA microarray method to examine the effects of clozapine on mouse brain function. After we injected clozapine into mice for 20 days, we decapitated the mice. We then used the DNA microarray method to examine the gene expression of mouse brain. We found some changes in the mouse brain treated with clozapine. For example, the intensity of a potassium channel spot decreased, and that of a serotonin receptor spot increased.

Gene expression profiles in the brain from phencyclidine-treated mouse by using DNA microarray.

Recently DNA microarray technology has been introduced into analyses of comprehensive biological functions. This DNA microarray is a new technology for simultaneous analysis to examine expression patterns of thousands of genes. It was thought that this technique should be very useful for examination of cellular and molecular mechanisms of drugs of abuse: cocaine, amphetamine, and others. This technology was therefore applied for the rapid analysis of gene expression in the brain from phencyclidine-treated mice. Mainly mouse DNA microarray was examined by using labeled cDNAs produced from a control mouse brain mRNA and from brain mRNA of mouse exposed to drugs as probes. Some changes in a probe from brain mRNA of drug-treated mouse could be observed, but it was necessary to examine another DNA microarray, including more samples from the brain.

The untranslated region of (mu)-opioid receptor mRNA contributes to reduced opioid sensitivity in CXBK mice.

It is well known that there are individual differences in a sensitivity to analgesics. Several lines of evidence have suggested that the level of opioid-induced analgesia is dependent on the level of expression of the mu-opioid receptor (mu-OR). However, the molecular mechanisms underlying the diversity of the level of the opioid receptor and the opioid sensitivity among individuals remain to be elucidated. In the present study, we analyzed the opioid-receptor genes of CXBK recombinant-inbred mice, which show reduced sensitivity to opioids. Northern blotting, nucleotide sequencing, and in situ hybridization histochemical analyses demonstrated that CXBK mice possessed mu-OR mRNA with a normal coding region but an abnormally long untranslated region (UTR). In addition, the mu-OR mRNA level in CXBK mice was less than in the control mice. Next, we produced littermate mice that had inherited two copies of the wild-type mu-OR gene, had inherited two copies of the CXBK mu-OR gene, and had inherited both copies of the mu-OR genes. In these mice, inheritance of the CXBK mu-OR gene was well correlated with less mu-OR mRNA and reduced opioid effects on nociception and locomotor activity. We conclude that the CXBK mu-OR gene is responsible for the CXBK phenotypes. Because UTR differences are known to affect the level of the corresponding mRNA and protein and because UTRs are more divergent among individuals than coding regions, the present findings suggest that opioid sensitivity may vary, depending on different mu-OR levels attributable to divergent UTR of mu-OR mRNA.

Primary malignant lymphoma of the brain: analysis of MMAC1 (PTEN) tumor suppressor gene.

With the use of RT-PCR (reverse transcriptase-polymerase chain reaction), Northern blot analysis, and Western blot analysis, seven primary brain lymphomas were examined for the state of the MMACI tumor suppressor gene. Nucleotide analysis of RT-PCR clones revealed no abnormality in the MMAC1 coding sequence in each case. Although Northern blot revealed variation among cases in the signal intensities for MMAC1 mRNA, Western blot revealed a distinct MMAC1 protein band in all cases, suggesting that the actual MMAC1 expressions were similar. In Western blot analysis of phosphorylated Akt (p-Akt), which is regulated positively by PI3K (phosphoinositide-3 kinase) and negatively by MMAC1, all the lymphomas revealed an Akt band but not a p-Akt band, suggesting that the MMAC1 phosphatase activity was maintained in each case. These findings suggest that the MMAC1 gene is normal in its coding sequence, gene expression, and phosphatase activity in the lymphomas. Thus, unlike the p16 and p15 tumor suppressor genes, which are frequently deleted and inactivated in brain lymphoma and represent a striking contrast to systemic lymphoma, MMAC1 may not play an important role in carcinogenesis in this tumor, as in the systemic counterpart.

The effects of DCG-IV and L-CCG-1 upon phencyclidine (PCP)-induced locomotion and behavioral changes in mice.

The behavioral changes of mice induced by acute and repeated i.p. injection of phencyclidine (PCP) were observed by measuring locomotor activity and stereotyped behavior. Then, the effects of metabotropic glutamate receptor (mGluR) agonists, DCG-IV and L-CCG-1, on the above behavioral changes induced by PCP were found. The effects of DCG-IV were very strong and completely depressed the PCP-induced hyperlocomotion. The effects of L-CCG-1 were not so strong. Repeated injection of PCP for 20 days into mice induced lower locomotor activity than that in acutely injected mice. These behavioral changes may be related with the negative symptoms of schizophrenia. In order to examine some molecular mechanisms of PCP-induced behavioral changes, Northern blot analysis of total RNA from prefrontal cortical tissues of mice treated with PCP, DCG-IV, and L-CCG-1 was carried out.

Involvement of G-protein-activated inwardly rectifying K (GIRK) channels in opioid-induced analgesia.

To investigate the role of G-protein-activated inwardly rectifying K+ (GIRK) channels in opioid-induced analgesia, we compared the effects of opioids in wild-type and weaver mutant mice having mutant GIRK channels. In the tail-flick and hot-plate tests, weaver mutant mice displayed significantly lower analgesia after either morphine or (-)-U-50488 administration. These findings suggest that GIRK channel activation is important in the induction of analgesia by opioids.

Molecular characterization of a novel gamma-glutamyl transpeptidase homologue found in rat brain.

A cDNA clone for a novel homologue to gamma-glutamyl transpeptidase (gamma-GTP), termed GTPH, was isolated from a rat brain expression cDNA library using antisera against total brain synaptosomal fractions. The cloned GTPH consists of 641 amino acid residues (78 kDa) and exhibits structural similarity with a conventional type of gamma-GTP that is predominantly expressed in the liver: They share significant amino acid homology (33% identity, 73% similarity) spanning over the entire sequence. RNA analyses revealed that GTPH mRNA expression is found only in the nervous system, including all brain regions, eyes and peripheral ganglia, and increases during development. Endogenous GTPH protein is a membrane-bound glycoenzyme and migrates as 90-100 kDa in polyacrylamide gels. Taken together, GTPH is a novel form of a gamma-GTP-like molecule expressed exclusively in the nervous system.

Demonstration of hydroxyindole-O-methyltransferase (HIOMT) mRNA expression in pineal parenchymal tumors: histochemical in situ hybridization.

The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors.

Cloning of a novel murine gene Sfmbt, Scm-related gene containing four mbt domains, structurally belonging to the Polycomb group of genes.

A novel cDNA clone encoding a protein structurally related to the transcriptional repressor Polycomb group (PcG) proteins, which regulate homeotic genes and others, was isolated from mouse and rat brain. The coding protein contained the SPM domain and mbt repeats, both of which are characteristic of the PcG proteins, and showed significant similarity in amino acid sequence to the Drosophila Sex comb on midleg (Scm) protein. Since this novel protein contains the mbt repeats in four tandem copies, we designated this murine gene as Sfmbt for Scm-related gene containing four mbt domains. Cloning and characterization of the mouse Sfmbt gene revealed that the coding sequence comprised 20 exons, dispersed along approximately 40kb, and mapped to the proximal part of Chromosome 14. Northern blot analysis showed that the Sfmbt mRNAs were expressed most abundantly in the adult testis, and less intensively in all other tissues examined.

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain.

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Inhibition by various antipsychotic drugs of the G-protein-activated inwardly rectifying K(+) (GIRK) channels expressed in xenopus oocytes.

To investigate the effects of various chemical classes of antipsychotic drugs: haloperidol, thioridazine, pimozide and clozapine, on the G-protein-activated inwardly rectifying K(+) (GIRK) channels, we carried out Xenopus oocyte functional assays with GIRK1 and GIRK2 mRNAs or GIRK1 and GIRK4 mRNAs. In oocytes co-injected with GIRK1 and GIRK2 mRNAs, application of each of the various antipsychotic drugs immediately caused a reduction of inward currents through the basally active GIRK channels. These responses were not observed in the presence of 3 mM Ba(2+), which blocks the GIRK channels. In addition, in uninjected oocytes, none of the drugs tested produced any significant current response. These results indicate that all the antipsychotic drugs tested inhibited the brain-type GIRK1/2 heteromultimeric channels. Furthermore, similar results were obtained in oocytes co-injected with GIRK1 and GIRK4 mRNAs, indicating that the antipsychotic drugs also inhibited the cardiac-type GIRK1/4 heteromultimeric channels. All the drugs tested inhibited, in a concentration-dependent manner, both types of GIRK channels with varying degrees of potency and effectiveness at micromolar concentrations. Only pimozide caused slight inhibition of these channels at nanomolar concentrations. We conclude that the various antipsychotic drugs acted as inhibitors at the brain-type and cardiac-type GIRK channels. Our results suggest that inhibition of both types of GIRK channels by these drugs underlies some of the side effects, in particular seizures and sinus tachycardia, observed in clinical practice.

Rare-type mutations of MMAC1 tumor suppressor gene in human glioma cell lines and their tumors of origin.

A total of 10 glioma cell lines were examined to evaluate the status of the MMAC1 gene, a candidate tumor suppressor gene. Six cell lines showed mutations with presumed loss of heterozygosity and 1 cell line showed no mRNA expression. The 6 mutations consisted of 3 3-bp deletions (codons 17, 101 or 199), 1 missense mutation (codon 252) and 2 truncation mutations (1 nonsense mutation at codon 233 and 12-bp insertion at codon 241). Among them, the 3-bp deletions, which are a rare type of mutation in MMAC1 gene, were located in the N-terminal half (codons 1-212) of the coding region, which is considered important in MMAC1 function. The missense mutation was located unusually in the C-terminal half (codons 212-403), but it was in a small region in which some other reported missense mutations are clustered. Thus, these 4 mutations were suggested to have functional effects on the MMAC1 activity, like the other 2 mutations with predicted protein truncations. By sequence analysis of cDNA clones, we confirmed that all the mutations including these 4 rare ones were in the MMAC1 gene, not in the PTH2 pseudogene. In 2 cases, we also examined the primary glioma tissues from which the cell lines had been derived and found the same mutations as in the cell lines in both cases. This suggested that the mutations in these cell lines were derived from the primary glioma tissues, but not from artifacts arising during long-term in vitro cultivation.

Ethanol opens G-protein-activated inwardly rectifying K+ channels.

Ethanol affects many functions of the brain and peripheral organs. Here we show that ethanol opens G-protein-activated, inwardly rectifying K + (GIRK) channels, which has important implications for inhibitory regulation of neuronal excitability and heart rate. At pharmacologically relevant concentrations, ethanol activated both brain-type GIRK1/2 and cardiac-type GIRK1/4 channels without interaction with G proteins or second messengers. Moreover, weaver mutant mice, which have a missense mutation in the GIRK2 channel, showed a loss of ethanol-induced analgesia. These results suggest that the GIRK channels in the brain and heart are important target sites for ethanol.

Unique behavioural phenotypes of recombinant-inbred CXBK mice: partial deficiency of sensitivity to mu- and kappa-agonists.

Recombinant-inbred CXBK mice have been used for various studies as putative mu-opioid-receptor deficient mice. However, CXBK mice have never been compared with gene-targeting mice lacking the mu-opioid receptor (muKO) and the K-opioid receptor (kappaKO). Here we report that CXBK mice show distinct behavioural phenotype in opioid-induced analgesia and sedation. Intraperitoneal (i.p.) administration of morphine (3 and 10 mg kg(-1)) induced significantly lower levels of analgesia in CXBK mice than in the control C57BL/6 mice, while higher doses of morphine (30 and 100 mg kg(-1)) induced marked analgesia in CXBK mice. CXBK mice also showed lower analgesia and sedation levels than did C57 mice after i.p. administration of U-50488 (10 and 30 mg kg(-1)). The partial deficiency of sensitivity to morphine and U-50488 of CXBK mice is in sharp contrast to the complete lack of sensitivity to morphine and U-50488 in muKO and kappaKO mice, respectively. Furthermore, CXBK mice showed a lower threshold for nociceptive stimuli when they were not given an opioid, suggesting that CXBK mice could have alterations in the genes related to the nociceptive threshold. These unique behavioural phenotypes of CXBK mice suggest unique genetic alterations in CXBK mice.

Establishment of two glioma cell lines from two surgical specimens obtained at different times from the same individual.

We established two glioma cell lines from two surgical specimens obtained at different times from the same patient. One (No. 9R), which was derived from the recurrent tumor (glioblastoma, grade IV), proliferated more rapidly in vitro than the other (No. 9) from the primary tumor (slightly anaplastic astrocytoma, grade II-III). No. 9R showed heterotransplantability in nude mice, whereas No. 9 did not. These findings indicate that No. 9R has a more aggressive or malignant nature than No. 9. Both cell lines showed homozygous deletion of the representative tumor suppressor p16 and p15 genes, but no p53 gene alteration. However, examination of the overall mRNA expression profile using a commercially available cDNA-spotted membrane revealed much higher expression levels of several mRNAs, at least, in No. 9R than in No. 9, although the relationship between these mRNAs and the growth potentials remained unknown. These two cell lines, derived from the same individual, with different proliferating potentials may be useful for studies on the molecular bases of glioma malignancy and progression.

14-3-3 protein eta chain gene (YWHAH) polymorphism and its genetic association with schizophrenia.

Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

Distinct regional distribution in the brain of messenger RNAs for the two isoforms of synaphin associated with the docking/fusion complex.

Synaphin is a 19,000 mol. wt cytosolic protein we first found to co-purify with the docking/fusion complex crucial to neurotransmitter release from presynaptic terminals. Two isoforms of synaphin (synaphins 1 and 2) (also called complexins II and I, respectively) exist in the rat brain. On density gradient centrifugation of a Triton X-100 extract of brain membranes, synaphin was found to be associated with the 7S complex that contains synaptotagmin, syntaxin, synaptosomal-associated protein of 25,000 mol. wt and vesicle-associated membrane protein. A smaller complex devoid of synaphins was also identified by immunoprecipitation with a monoclonal antibody against synaptosomal-associated protein of 25,000 mol. wt. Messenger RNAs for synaphins 1 and 2 were expressed predominantly in the brain. In situ hybridization using probes specific to synaphins 1 and 2 indicated that the distribution of their mRNAs was significantly different in brain regions such as olfactory bulb, hippocampus, cerebral cortex, piriform cortex, cerebellum, thalamus and facial nuclei. These results show synaphin as a component of the 7S complex and suggest different physiological implications for the two isoforms.

Role of nitric oxide in pathogenesis underlying ischemic cerebral damage.

1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia. 2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage. 3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia. 4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.

Distribution of prepro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems.

Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development.