Toshiyuki Araki

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.

Involvement of p53 in DNA Strand Break‐induced Apoptosis in Postmitotic CNS Neurons

The tumour suppressor p53 gene serves as a critical regulator of the cell cycle and of apoptosis following the exposure of normal cells to DNA damage. To examine the role of p53 in postmitotic CNS neurons, we cultured cerebellar neurons from normal wild‐type mice and mutant p53‐null mice under various conditions inducing neuronal death. When cerebellar neurons from 15‐ to 16‐day postnatal wild‐type mice were treated with ionizing radiation or DNA‐damaging agents, massive neuron death occurred after 24‐72 h. In contrast, neurons from p53+ mice evidently resisted γ‐irradiation and some DNA‐damaging agents, such as etoposide and bleomycin. On the other hand, low‐K+ medium‐induced apoptosis of cerebellar neurons was not affected by p53 status. Neither cell cycle progression nor DNA synthesis occurred during cell death induced by γ‐irradiation and low‐K+ medium, as well as in normal cultures of p53+/+ and p53‐/‐ neurons. These results suggest that p53 is required for the apoptotic death of postmitotic cerebellar neurons induced by DNA strand breaks.

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.

Region-specific expression of GABAA receptor α3 and α4 subunits mRNAs in the rat brain

Abstract The expression of mRNAs encoding the α3 and α4 subunit of the γ-aminobutyric acid A (GABAA) receptor in the rat brain was investigated by in situ hybridization histochemistry. Both subunits showed a wide but uneven distribution, which did not coincide with the distribution of any other subunit so far reported. The cerebral cortex, anterior olfactory nucleus, lateral septum, subiculum, lateral and medial nuclei of the amygdaloid complex, anterior nuclei of the thalamus, pars compacta of the substantia nigra, trigeminal sensory nuclei, and cochlear nucleus were some of the areas where strong expression of mRNA for both the α3 and α4 subunits was detected. In the granular cell layer of the olfactory bulb, caudate-putamen, tenia tecta, pyramidal cell layer of the CA region and granular cell layer of the dentate gyrus in the hippocampal formation, dorsomedial and ventrolateral nuclei of the thalamus, dorsal part of the lateral geniculate body, preolivary nuclei and pontine nuclei, only the α4 subunit showed strong expression. The diverse distribution of these two subunits is considered to indicate that each has a different role in the central nervous system.

Localization of GABAA-receptor γ2-subunit mRNA-containing neurons in the rat central nervous system

The localization of neurons containing mRNA of the gamma 2-subunit of GABAA receptors was examined in the rat central nervous system with in situ hybridization histochemistry using an oligonucleotide probe to the sequence of the gamma 2-subunit. Neurons containing the gamma 2-subunit mRNA were widely but unevenly distributed in the brain. The location of gamma 2-subunit mRNA-containing neurons differed from those containing alpha- or beta-subunits. According to our results brain regions can be divided into three categories: one containing only gamma 2-subunit, one containing mRNA of at least one subunit other than gamma 2 and one containing more than one other subunit but not the gamma 2-subunit. The distribution of strongly labeled nuclei partly coincided with that of glutamate decarboxylase, suggesting that the GABAA receptor gamma 2-subunit would be involved in an autoreceptive mechanism of the GABAergic transmission.

GABAA Receptor subunit messenger RNAs show differential expression during cortical development in the rat brain

Developmental changes of the expression of various GABAA receptor subunits (alpha 1, alpha 3, alpha 4, beta 1-3, and gamma 2) were examined in the fetal rat cerebral cortex using in situ hybridization histochemistry. The subunits showed three main patterns of development. The alpha 1 subunit showed the first pattern, in which no expression was observed during embryonic development. The alpha 4 and beta 1 subunits showed the second pattern, in which expression was observed in both the undifferentiated neuroepithelium and the developing cortical layers. The alpha 3, beta 2, beta 3, and gamma 2 subunits showed the third pattern, in which expression was only seen in the developing cortical layers. These findings strongly suggest the following: (i) the alpha 1 subunit is involved in GABAergic transmission in the mature cerebral cortex; (ii) the alpha 4 and beta 1 subunits are involved in both the differentiation of the neuroepithelium and the development of the cortical plate, and (iii) the alpha 3, beta 2, beta 3, and gamma 2 subunits are involved in the development of the cortical plate. Subunits already expressed on embryonic day 13 (beta 1, beta 3, and gamma 2) appear especially likely to have a special role in neuronal development.

The GABAA receptor γ1 subunit is expressed by distinct neuronal populations

The distribution of GABAA receptor gamma 1 subunit was examined in the rat central nervous system using in situ hybridization histochemistry. The gamma 1 subunit was expressed in relatively limited areas compared to other subunits investigated previously. The brain regions strongly expressing this subunit were the septum, globus pallidus, bed nucleus of the stria terminalis, hypothalamic periventricular nucleus, supraoptic nucleus, medial and central nuclei of the amygdaloid complex, medial part of the medial geniculate body, substantia nigra pars reticulata, interpeduncular nucleus, lateral parabrachial nucleus, Purkinje cell layer of the cerebellum, and inferior olivary nucleus. This relatively limited expression implies a possible role of gamma 1 subunit in relation to some specific neuronal circuit.

Changes in c-Jun but not Bcl-2 family proteins in p53-dependent apoptosis of mouse cerebellar granule neurons induced by DNA damaging agent bleomycin

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.

BIT/SHPS-1 enhances brain-derived neurotrophic factor-promoted neuronal survival in cultured cerebral cortical neurons.

Abstract: Brain‐derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS‐1 (brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/SHP substrate 1) is a substrate of Shp‐2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS‐1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS‐1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS‐1 were replaced with phenylalanine. Overexpression of wild‐type BIT/SHPS‐1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/SHPS‐1 itself and an association of Shp‐2 with BIT/SHPS‐1 even without addition of BDNF. We found that BDNF‐promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS‐1 does not depend on its tyrosine phosphorylation. BDNF‐induced activation of mitogen‐activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF‐induced activation of Akt was enhanced in neurons expressing wild‐type or 4F mutant BIT/SHPS‐1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3‐kinase, blocked the enhancement of BDNF‐promoted neuronal survival in both neurons expressing wild‐type and 4F mutant BIT/SHPS‐1. These results indicate that BIT/SHPS‐1 contributes to BDNF‐promoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3‐kinase‐Akt pathway. Our results suggest that a novel action of BIT/SHPS‐1 does not occur through tyrosine phosphorylation of BIT/SHPS‐1 in cultured cerebral cortical neurons.

Shp‐2 Specifically Regulates Several Tyrosine‐Phosphorylated Proteins in Brain‐Derived Neurotrophic Factor Signal

Abstract: Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival and regulates plasticity of various types of neurons. BDNF binds to TrkB, a receptor tyrosine kinase, which results in the activation of a variety of signaling molecules to exert the various functions of BDNF. Shp‐2, a Src homology 2 domain‐containing cytoplasmic tyrosine phosphatase, is involved in neurotrophin signaling in PC12 cells and cultured cerebral cortical neurons. To examine the roles of Shp‐2 in BDNF signaling in cultured rat cerebral cortical neurons, the wild‐type and phosphatase‐inactive mutant (C/S mutant) forms of Shp‐2 were ectopically expressed in cultured neurons using recombinant adenovirus vectors. We found that several proteins tyrosinephosphorylated in response to BDNF showed enhanced levels of tyrosine phosphorylation in cultured neurons infected with C/S mutant adenovirus in comparison with those infected with the wild‐type Shp‐2 adenovirus. In addition, in immunoprecipitates with anti‐Shp‐2 antibody, we also observed at least four proteins that displayed enhanced phosphorylation in response to BDNF in cultured neurons infected with the C/S mutant adenovirus. We found that the Shp‐2‐binding protein, brain immunoglobulin‐like molecule with tyrosine‐based activation motifs (BIT), was strongly tyrosine‐phosphorylated in response to BDNF in cultured neurons expressing the C/S mutant of Shp‐2. In contrast, the level of BDNF‐induced phosphorylation of mitogen‐activated protein kinase and coprecipitated proteins with anti‐Trk and Grb2 antibodies did not show any difference between neurons infected with these two types of Shp‐2. Furthermore, the survival effect of BDNF was enhanced by the wild type of Shp‐2, although it was not influenced by the C/S mutant of Shp‐2. These results indicated that in cultured cerebral cortical neurons Shp‐2 is specifically involved in the regulation of several tyrosine‐phosphorylated proteins, including BIT, in the BDNF signaling pathway. In addition, the phosphatase Shp‐2 may not influence the level of BDNF‐induced activation of mitogen‐activated protein kinase in cultured cortical neurons. Further, Shp‐2 may have potential to positively regulate BDNF‐promoting neuronal survival.