Akio Wanaka

Localization of mRNA for c-kit receptor and its ligand in the brain of adult rats: an analysis using in situ hybridization histochemistry.

Localization of mRNA for the c-kit receptor and its ligand (Sl factor) in the brain of adult rats was studied using in situ hybridization histochemistry. The mRNA for the c-kit receptor was detected in the forebrain, the lower brain stem and the cerebellum. In the forebrain, the c-kit mRNA signals were detected in the olfactory bulb, the caudate-putamen, throughout the superficial cortex, the accumbens nucleus, the nucleus of vertical limb diagonal band, the bed nucleus of anterior commissure, Ammons horn, the entopeduncular nucleus, the subthalamic nucleus, the dorsal raphe nucleus, the parasubiculum, the presubiculum, the ventricular nucleus of lateral lemniscus, and the entorhinal cortex. In the lower brain stem, the signals were detected in the inferior colliculus, the spinal vestibular nucleus, the spinal tract nucleus of trigeminal nerve, and the pyramidal tract. In the cerebellum, the signals were detected in the molecular layer of the cortex and cerebellar nuclei. By contrast, the signals of mRNA for Sl factor were detected in the forebrain and the cerebellum. In the forebrain, the signals were detected in the olfactory bulb, the endopiriform nucleus, the septohippocampal nucleus, the habenular nuclei, and most of the thalamic nuclei. In the cerebellum, the signals were detected in Purkinje cells. Several pairs of structures were found in which mRNA of either the c-kit receptor or the Sl factor was expressed and between which the synaptic connection had been reported, suggesting that the interaction between the c-kit receptor and the Sl factor may play some roles in the development of such synaptic connections.

Localization and functional coupling of HGF and c-Met/HGF receptor in rat brain : implication as neurotrophic factor

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, has mitogenic, motogenic and morphogenic activities for various cell types and functions as a organotrophic factor for regeneration of the liver, kidney and lung. We obtained evidence that HGF may function as a novel neurotrophic factor in the central nervous system. Northern blot analysis showed that 6 kb HGF mRNA and 9 kb c-Met/HGF receptor mRNA are expressed in various regions of the adult rat brain. In situ hybridization analysis revealed that intense hybridization signals for HGF mRNA were localized in cerebral cortex, hippocampus and amygdala. Consistently, specific localization of HGF protein in neurons of these regions was detected by immunohistochemical analysis and non-neuronal glial cells in cingulum, cerebellum, pons and medulla were also specifically stained. Specific intense hybridization signals for c-Met/HGF receptor mRNA were also widely distributed in the brain, including neurons of olfactory bulb, cerebral cortex, primary olfactory cortex, hippocampus and cerebellum. On the basis of the co-expression of HGF and c-Met/HGF receptor in hippocampal neurons, we found that HGF prolonged survival of embryonic hippocampal neurons in primary culture: HGF elicited maximal surviving effect at 0.5-1 ng/ml and the potency was comparable to that of nerve growth factor. More importantly, expression of both HGF and c-Met/HGF receptor mRNAs was markedly induced in response to cerebral ischemic injury. We propose that HGF functions as a neurotrophic factor in the central nervous system and that this neurotrophic function may have a role in the survival and reconstruction of specific neurons in response to cerebral injury.

Molecular Cloning of a Novel Polypeptide, DP5, Induced during Programmed Neuronal Death

To study the molecular mechanisms underlying neuronal programmed cell death (PCD), we performed differential display screening for genes, the expression of which was induced during PCD in the sympathetic neuron culture model deprived of NGF. We cloned a gene encoding a novel polypeptide (DP5) which consisted of 92 amino acids. DP5 polypeptide had no homology with any other known protein and contained no motif that would indicate its putative biochemical functions. DP5 mRNA levels peaked at 15 h after nerve growth factor withdrawal, concurrent with the time at which neurons were committed to die. The induction of DP5 gene expression was blocked when cell death was rescued by treatment with cycloheximide, KCl, or the cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in cultured sympathetic neurons was in itself sufficient to induce apoptosis. These results suggest that DP5 plays a role in programmed neuronal death.

Localization of glycine receptors in the rat central nervous system: An immunocytochemical analysis using monoclonal antibody

The localization of glycine receptors was immunocytochemically examined in the rat brain using a monoclonal antibody against the affinity-purified glycine receptor. Glycine receptors were concentrated in the lower brainstem, whereas no immunoreactivity was observed in the diencephalon and forebrain except in a few diencephalic nuclei. The highest density of receptors was found in the cranial motor nuclei, reticular formation, parabrachial area, dorsal and ventral cochlear nuclei, and dorsal and ventral tegmental nuclei. Differences were observed in the distribution of immunoreactive elements in the various brain regions. In the cerebellar cortex, the immunoreactivity was exclusively seen along the dendrites of the Purkinje cells. On the other hand, glycine receptors were detected on the cellular membrane of the soma of the cochlear nuclei, trigeminal motor nucleus, parabrachial area, lateral reticular nucleus, dorsal nucleus of the lateral lemniscus, cerebellar nuclei, trigeminal spinal nucleus, anterior horn and reticular formation. In other regions, the receptors were evenly distributed throughout the neuropil.

Differential expression of two members of FGF receptor gene family, FGFR-1 and FGFR-2 mRNA, in the adult rat central nervous system

The fibroblast growth factor (FGF) receptor gene family now contains four members that encode homologous receptor-tyrosine kinases (RTKs) to each other. We have demonstrated that one of the members, FGFR-1 (also termed as flg), is expressed in the widespread but specific neuronal populations in the adult rat central nervous system (CNS). In the present study, we examined the expression pattern of another member, FGFR-2 (also termed as bek) and compared it with that of FGFR-1. In contrast with FGFR-1, we detected FGFR-2 expression primarily in the fiber tracts of the adult rat CNS, suggesting that the oligodendrocytes are the main sites of the FGFR-2 expression. These findings indicate that FGF may regulate neurons and glial cells through different subtypes of its cognate receptor.

DIFFERENTIAL EXPRESSION OF SGK MRNA, A MEMBER OF THE SER/THR PROTEIN KINASE GENE FAMILY, IN RAT BRAIN AFTER CNS INJURY

We cloned genes the expression of which were induced 3 days after cortical injury of rat brain by a differential display technique, and four novel and known sequences were isolated. Among these sequences, the sgk gene which was recently identified as a novel member of the serine/threonine protein kinase gene family, was selected for analysis of its expression patterns in rat brain by northern blotting and in situ hybridization, because hybridization signals were strong at the lesion sites. Expression of sgk mRNA was induced within 3 days after injury, and was maintained at a high level for at least 14 days. The cells which strongly expressed the sgk gene were in the deep layers of the cortex and in the corpus callosum. In situ hybridization analysis for sgk and myelin proteolipid protein mRNA using serial sections showed that the distribution of both signals was very similar at the damaged regions. Therefore, it is likely that the sgk transcript is expressed by oligodendrocytes after brain injury. Investigation of the developmental expression of the sgk gene showed that neurons in layers I and II of the cortex, lateroposterior and laterodorsal thalamic nucleus, and ventral posterolateral and posteromedial thalamic nucleus strongly expressed sgk mRNA at postnatal day 1 and day 7, but these neurons showed no expression in fetal or adult brain. These results suggest that the induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain.

Specific expressions of Fyn and Lyn, lymphocyte antigen receptor-associated tyrosine kinases, in the central nervous system.

The Src-like protein-tyrosine kinases Fyn and Lyn are expressed in lymphocytes. Fyn is expressed in T cells at elevated levels and is associated with the T cell antigen receptor complex, whereas Lyn is expressed in B cells and is associated with membrane-bound immunoglobulin. Thus, these kinases are suggested to participate in antigen-mediated signal transduction in lymphocytes. Previous report showed that fyn was also expressed in brain, but its cellular distribution was not examined. Expression of Lyn in neural tissues was not previously reported. Here we report that both fyn and lyn are expressed in discrete regions of the brain. To throw light on their functions in the brain, we investigated their expressions during brain ontogenesis in mice. In situ hybridization analysis showed that Fyn mRNA was specifically expressed in neurons of embryos and newborn mice. In adult animals, fyn mRNA was expressed in oligodendrocytes as well as neurons. In contrast, the expression of lyn mRNA was relatively low in brains of embryos and newborn mice, but in adults the transcript was specifically expressed in the granular layer of the cerebellum. Therefore, the Fyn and Lyn kinases may regulate distinct functions of specific cells during brain development. The specific expressions of Fyn and Lyn in both lymphatic and neural tissues could suggest common signalling mechanisms in the immune system and central nervous system.

Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate.

SummaryWe found that large cells in the dorsal root and trigeminal ganglia contained glutamate-like immunoreactivity. Immunoreactive neurons were not detected in the superior cervical or pterygopalatine ganglia. These findings indicated that glutamate is a neurotransmitter or neuromodulator of large cells of sensory ganglia.

The Cell Death-promoting Gene DP5, Which Interacts with the BCL2 Family, Is Induced during Neuronal Apoptosis Following Exposure to Amyloid β Protein

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. Previously, we reported that DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. Here, we analyzed DP5 gene expression and the specific interaction of DP5 with Bcl-xl during neuronal death induced by amyloid-β protein (A β). DP5 mRNA was induced 6 h after treatment with A β in cultured rat cortical neurons. The protein encoded by DP5 mRNA showed a specific interaction with Bcl-xl. Induction of DP5 gene expression was blocked by nifedipine, an inhibitor of l-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A β. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A β, and its binding could impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A β.

MFH-1, a new member of the fork head domain family, is expressed in developing mesenchyme.

We have isolated a novel mouse gene, MFH‐1 (mesenchyme fork head 1) that is related to the Drosophila fork head and rat HNF3 genes. MFH‐1 encodes a distinct fork head domain that is classified into a distinct subfamily. A recombinant MFH‐1 protein could bind to the HNF3 binding site. MFH‐1 is expressed temporally in developing embryos, first in the non‐notochordal mesoderm and later in areas of mesenchymal condensation in the trunk, head, and limbs. Our results suggest that MFH‐1 might be involved in the formation of special mesenchymal tissues.