Kazuhiro Kitada

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-κb-inducing kinase

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyers patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT β-receptor (Ltβr, encoded by Ltbr ) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 (ref. 11) is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-κb-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-κb activation induced by an excess of Ltβr. Our observations agree with previous reports that Ltβr-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-κb activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6; ref. 13), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr ( ref. 12), Cd40 (Refs 14, 15), Cd30 (Refs 16, 17) and Ltβr (refs 18, 19, 20, 21). The phenotypes of aly/aly mice are more severe than those of Ltbr–/– mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

Cblb is a major susceptibility gene for rat type 1 diabetes mellitus

The autoimmune disease type 1 diabetes mellitus (insulin-dependent diabetes mellitus, IDDM) has a multifactorial etiology. So far, the major histocompatibility complex (MHC) is the only major susceptibility locus that has been identified for this disease and its animal models. The Komeda diabetes-prone (KDP) rat is a spontaneous animal model of human type 1 diabetes in which the major susceptibility locus Iddm/kdp1 accounts, in combination with MHC, for most of the genetic predisposition to diabetes. Here we report the positional cloning of Iddm/kdp1 and identify a nonsense mutation in Cblb, a member of the Cbl/Sli family of ubiquitin-protein ligases. Lymphocytes of the KDP rat infiltrate into pancreatic islets and several tissues including thyroid gland and kidney, indicating autoimmunity. Similar findings in Cblb-deficient mice are caused by enhanced T-cell activation. Transgenic complementation with wildtype Cblb significantly suppresses development of the KDP phenotype. Thus, Cblb functions as a negative regulator of autoimmunity and Cblb is a major susceptibility gene for type 1 diabetes in the rat. Impairment of the Cblb signaling pathway may contribute to human autoimmune diseases, including type 1 diabetes.

Accumulation of N-Acetyl-L-Aspartate in the Brain of the Tremor Rat, a Mutant Exhibiting Absence-Like Seizure and Spongiform Degeneration in the Central Nervous System

Abstract: The tremor rat is a mutant that exhibits absence‐like seizure and spongiform degeneration in the CNS. By positional cloning, a genomic deletion was found within the critical region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N‐acetyl‐L‐aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4‐ to 10‐Hz polyspikes or spikewave‐like complexes in cortical and hippocampal EEG, concomitantly with behavior characterized by sudden immobility and staring. These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly.

Transposon-tagged mutagenesis in the rat.

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.

Correlation between genetic and cytogenetic maps of the rat

To correlate rat genetic linkage maps with cytogenetic maps, we localized 25 new cosmid-derived simple sequence length polymorphism (SSLP) markers and 14 existing genetic markers on cytogenetic bands of chromosomes, using fluorescence in situ hybridization (FISH). Next, a total of 58 anchor loci, consisting of the 39 new and 19 previously reported ones, were integrated into the genetic linkage maps. Since most of the new anchor loci were developed to be localized near the terminals of the genetic or cytogenetic maps for each chromosome, the orientation and coverage of the whole genetic linkage maps were determined or confirmed with respect to the cytogenetic maps. Thus, we provide here a new base for rat genetic maps.

Epileptic seizures induced by N-acetyl-l-aspartate in rats: in vivo and in vitro studies

Tremor rat (tm/tm), the parent strain of spontaneously epileptic rat (SER: zi/zi, tm/tm), exhibits absence-like seizures characterized by 5-7 Hz spike-wave-like complexes on cortical and hippocampal electroencephalograms (EEG) after 10 weeks of age, prior to development of convulsive seizures. Recently, this animal model has been demonstrated to display a genomic microdeletion within the critical region of tm, where aspartoacylase hydrolyzing N-acetyl-L aspartate (NAA) is located, besides showing the ability to accumulate NAA in the brain. Therefore, the present study was performed to determine the involvement of NAA in the induction of epileptic seizures. When NAA (4 micromol) was applied intracerebroventricularly (i.c.v.) to normal Wistar rats, 4-10 Hz polyspikes and/or spike-wave-like complexes followed by absence-like seizure before persistent 1-5 Hz waxing high-voltage after-discharges were observed on cortical and hippocampal EEG. At a higher dose (8 micromol), NAA induced convulsive seizures. The absence-like seizures with polyspikes and/or spike-wave-like complexes on the EEG were also observed with i.c.v. NAA in premature tremor rats without seizures. The NAA-induced seizures in normal rats were antagonized by i.c.v. glutamic acid diethyl ester, a non-selective glutamate receptor antagonist. In addition, NAA applied to the bath rapidly induced a long-lasting depolarization concomitantly with repetitive firings in hippocampal CA3 neurons of normal rat brain slice preparations. These findings suggest that NAA is involved in the induction of absence-like seizures and/or convulsion, probably via glutamate receptors.

Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat. In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER. Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed. After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER. The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector. Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration. No inhibitory effects were observed 10 and 14 days after AxASPA administration. In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER. Survival rates did not differ between AxASPA- and AxLacZ-treated SERs. Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.

A Mutation in the Gene Encoding Mitochondrial Mg2+ Channel MRS2 Results in Demyelination in the Rat

The rat demyelination (dmy) mutation serves as a unique model system to investigate the maintenance of myelin, because it provokes severe myelin breakdown in the central nervous system (CNS) after normal postnatal completion of myelination. Here, we report the molecular characterization of this mutation and discuss the possible pathomechanisms underlying demyelination. By positional cloning, we found that a G-to-A transition, 177 bp downstream of exon 3 of the Mrs2 (MRS2 magnesium homeostasis factor (Saccharomyces cerevisiae)) gene, generated a novel splice acceptor site which resulted in functional inactivation of the mutant allele. Transgenic rescue with wild-type Mrs2-cDNA validated our findings. Mrs2 encodes an essential component of the major Mg2+ influx system in mitochondria of yeast as well as human cells. We showed that the dmy/dmy rats have major mitochondrial deficits with a markedly elevated lactic acid concentration in the cerebrospinal fluid, a 60% reduction in ATP, and increased numbers of mitochondria in the swollen cytoplasm of oligodendrocytes. MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was dominantly expressed in neurons rather than oligodendrocytes and was ultrastructurally observed in the inner membrane of mitochondria. Our observations led to the conclusion that dmy/dmy rats suffer from a mitochondrial disease and that the maintenance of myelin has a different mechanism from its initial production. They also established that Mg2+ homeostasis in CNS mitochondria is essential for the maintenance of myelin.

The Myelin Vacuolation ( mv ) Rat with a Null Mutation in the Attractin Gene

We recently found a spontaneous tremor mutant in an outbred colony of Sprague-Dawley rats. The tremor behavior was exhibited from around 3 weeks of age and inherited as an autosomal recessive trait. The mutant rats had variously sized vacuoles in the neuropil and white matter throughout the central nervous system, especially in the brain stem, cerebellum, and spinal cord. Ultrastructurally these vacuoles mainly consisted of splitting of myelin lamella both in the periaxonal and intermyelinic spaces. Linkage analysis using intercross progeny between the myelin vacuolation (mv) rat, named after the pathologic characteristics, and normal control rat strains showed that the mv phenotypes were cosegregated with polymorphic markers adjacent to the Atrn (Attractin, formerly zi [zitter]) locus on rat chromosome 3. A test for allelism suggested that the mv mutation was a new allele in Atrn. In comparison with a marked decrease of Atrnzi/Arnzi, Northern blot analysis revealed no expression of Atrn mRNA in the brain of the mv rats. Finally, a genomic deletion including exon 1 of the mv rats was detected by genomic and sequence analyses. Discovery of the rat null mutation Atrnmv, different from Atrnzi, provides a new animal model for studying the functions of the attractin protein.

Cloning of the rat steroid sulfatase gene (Sts), a non-pseudoautosomal X-linked gene that undergoes X inactivation.

Although the human steroid sulfatase (STS) gene has been cloned and characterized in detail, several attempts to clone its mouse homologue, with either anti-human STS antibodies or human STS cDNA probes, have failed, suggesting a substantial divergence between these genes. However, partial amino-terminal sequence from purified rat liver STS is very similar to its human counterpart, and sequence comparisons have revealed several domains that are conserved among all the sulfatases characterized to date. Thus, we used a degenerate-primer RT-PCR approach to amplify a 321-bp fragment from rat liver cDNA, which was used as a probe to clone and characterize the complete cDNA. Comparison of the protein coding region between the rat and human genes showed 66% homology both at the DNA and the protein levels. STS activity was conferred to STS(-) A9 cells upon transfection with a rat Sts expression construct, indicating the authenticity of the cloned cDNA. While Sts has been shown to be located in the mouse pseudoautosomal region, both physical and genetic mapping demonstrate that Sts is not pseudoautosomal in the rat. The overall genomic organization of rat Sts and human STS is very similar, except that the insertion site for intron 1 in the rat is 26 bp upstream from that in the human. Rat Sts is only 8.2 kb long, while the human STS spans over 146 kb.