Kazuki Nakao

Loss of teratogenic response to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in mice lacking the Ah (dioxin) receptor

The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand‐activated transcription factor that is considered to mediate pleiotropic biological responses such as teratogenesis, tumour promotion, epithelial hyperplasia and the induction of drug‐metabolizing enzymes to environmental contaminants usually represented by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). In contrast to the role of AhR in the regulatory mechanism of xenobiotic‐metabolizing enzymes, there is no direct proof that the AhR is involved in the teratogenic effects of TCDD.

Specific deficit of the ON response in visual transmission by targeted disruption of the mGIuR6 gene

Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber—CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber—CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber—CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

K-Ras is essential for the development of the mouse embryo

ras genes encode members of the small GTP-binding proteins. Ras protein is highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice

To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (KanR) and the rpsL+ gene (strAS) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (KmS) and streptomycin-resistant (SmR) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to KmR and SmS, since both the KanR and rpsL genes exhibited dominant traits of KmR and SmS, respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both KmR and SmR. Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10(-5). In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.

CHARACTERIZATION OF EXCITATORY AMINO ACID NEUROTOXICITY IN N-METHYL-D-ASPARTATE RECEPTOR-DEFICIENT MOUSE CORTICAL NEURONAL CELLS

Roles and mechanisms of N‐methyl‐D‐aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor‐deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild‐type NR1+/+, heterozygous NR1+/‐ and homozygous mutant NR1‐/‐ mice at 15–17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1+/+ and NR1+/‐ neuronal cells to glutamate or NMDA, but not kainate or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1‐/‐ cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effectivein inducing the death of NR1+/+ and NR1+/‐ neuronal cells after prolonged exposure (20–24 h). However, NMDA had no neurotoxic effects on NR1‐/‐ cells, although the other three compounds were neurotoxic with potencies comparable to those for NR1+/+ and NR1+/‐ cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1+/+ and NR1‐/‐ neuronal cells to NMDA selectively killed the NMDA receptor‐expressing cells without any appreciable effects on neighbouring NMDA receptor‐deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA‐evoked neuronal cell death.