Hiroshi Kiyama

mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells

ABSTRACT TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis

The localization of calcitonin gene-related peptide (CGRP) and substance P (SP) in the rat trigeminal ganglion was examined by means of the indirect immunofluorescent method. About 40% of neurons in the ganglion contained CGRP-like immunoreactivity (CGRPI), while about 20% of neurons showed SP-like immunoreactivity (SPI). In serial sections, nearly all the SPI neurons contained CGRPI.

The differential expression patterns of messenger RNAs encoding non-N-methyl-d-aspartate glutamate receptor subunits (GluR1–4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.

Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation

Activating transcription factor 3 (ATF3) is induced and functions both as a cellular response to stress and to stimulate proliferation in multiple tissues. However, in the nervous system ATF3 is expressed only in injured neurons. Here we reveal a function of ATF3 in neurons under death stress. Overexpression of ATF3 by adenovirus inhibits the mitogen-activated kinase kinase kinase 1 (MEKK1)–c-Jun N-Terminal Kinase (JNK)-induced apoptosis and induces neurite elongation via Akt activation in PC12 cells and superior nerve ganglion neurons. A DNA microarray study reveals that ATF3 expression and JNK activation induce expression of the heat shock protein 27 (Hsp27). Immunoprecipitation analysis and promoter assay for Hsp27 expression suggest that both ATF3 and c-Jun are necessary for transcriptional activation of Hsp27. Hsp27 expression significantly inhibits JNK-induced apoptosis as well as Akt activation in PC12 cells and superior cervical ganglion neurons. We conclude that the combination of ATF3 and c-Jun induces the anti-apoptotic factor Hsp27, which directly or indirectly activates Akt, and thereby possibly inhibits apoptosis and induces nerve elongation. Our results suggest that ATF3- and c-Jun-induced Hsp27 expression is a novel survival response in neurons under death stress such as nerve injury.

Vascular endothelial growth factor rescues hippocampal neurons from glutamate-induced toxicity: signal transduction cascades

Vascular endothelial growth factor (VEGF) is known as a selective endothelial cell mitogen that promotes angiogenesis and increases blood vessel formation in vivo. Here we report that VEGF has protective effects on primary hippocampal neurons against glutamate toxicity by acting on phosphatidylinositol 3‐kinase (PI3‐K)/Akt pathways and mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathways, operating independently of one another. Decrease in the VEGFs neuroprotective effect resulting from inhibition of either pathway alone was significantly enhanced by simultaneous inhibition of both pathways. However, adenovirus‐mediated expression of either the active form of Akt or of MEK significantly inhibited glutamate‐induced neuronal death. Treatment with antisense ODN against Flk‐1, but not against Flt‐1, blocked the effect of VEGF on the activation of Akt and ERK and glutamate‐induced neuronal death. These findings suggest that VEGF has a protective effect on hippocampal neurons against glutamate‐induced toxicity and that this effect is dependent on PI3‐ K/Akt and MEK/ERK signaling pathways mediated primarily through Flk‐1 receptor.

Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53.

Survival factors suppress apoptosis by activating the serine/threonine kinase Akt. To investigate the molecular mechanism underlying activated Akts ability to protect neurons from hypoxia or nitric oxide (NO) toxicity, we focused on the apoptosis-related functions of p53 and caspases. We eliminated p53 by employing p53-deficient neurons and increased p53 by infection with recombinant adenovirus capable of transducing p53 expression, and we now show that p53 is implicated in the apoptosis induced by hypoxia or NO treatments of primary cultured hippocampal neurons. Although hypoxia and NO induced p53, treatment with insulin-like growth factor-1 significantly inhibited caspase-3-like activation, neuronal death and transcriptional activity of p53. These insulin-like growth factor-1 effects are prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. Adenovirus-mediated expression of activated-Akt kinase suppressed p53-dependent transcriptional activation of responsive genes such as Bax, suppressed caspase-3-like protease activity and suppressed neuronal cell death with no effect on the cellular accumulation and nuclear translocation of p53. In contrast, overexpression of kinase-defective Akt failed to suppress these same activities. These results suggest a mechanism where Akt kinase activation reduces p53s transcriptional activity that ultimately rescues neurons from hypoxia- or NO-mediated cell death.

A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry

Using in situ hybridization histochemistry, we examined the biosynthesis of the preprotachykinin-A (PPTA) mRNAs, which code for substance P (SP) and also a related peptide, substance K (SK), in the rat dorsal root ganglion (DRG). The animals were injected with a small amount of formalin in the right hindpaw for nociceptive chemical stimulation. Analysis of hybridization signals in the emulsion autoradiography showed that the lumbar-5 DRG neurons expressing PPTA mRNAs were significantly increased in number and signal intensity on the formalin-injected side. Such elevation of the PPTA mRNA expression in the DRG was observed as early as 3 h after formalin injection. These results show that the expression of the PPTA gene is activated by chemogenic noxious stimuli.

Region-specific expression of subunits of ionotropic glutamate receptors (AMPA-type, KA-type and NMDA receptors) in the rat spinal cord with special reference to nociception

The present study attempted to explore the gene expression of the subunits (GluR1-4) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor, subunit (GluR5) of kainic acid (KA)-type receptor, NR1 [a subunit of N-methyl-D-aspartate (NMDA) receptors] and the possible glutamate-binding subunit of an NMDA receptor complex in the dorsal horn of the rat spinal cords using in situ hybridization histochemistry. These results were compared with those of the spinal motor neurons. Expression of the subunits of the AMPA-type receptor was also examined at the protein level using immunocytochemistry, with reference to the motor neurons. Although all the four subunits of the AMPA-type receptor were expressed throughout the dorsal horn, the pattern of expression was different according to the dorsal horn region and to the subunits. GluR2 showed the strongest expression in the dorsal horn. Huge numbers of strongly labelled cells formed a dense collection in lamina II and superficial parts of lamina III. Many neurons in lamina II and superficial parts of lamina III expressed GluR1 moderately. Scattered neurons moderately expressing GluR3 were also seen in these regions, while the expression of GluR4 was very low. Labelling of the dorsal horn neurons by the GluR5 probe was low, and NR1 probe and a glutamate-binding subunit of an NMDA receptor complex probe labelled them diffusely with low to moderate intensity. These findings show a close relationship between the glutamergic nociceptive primary afferent system and AMPA-type receptors in which GluR2 is especially highly expressed. The present study further showed that the expression pattern of the glutamate receptors in the spinal sensory neurons differs considerably from that of spinal motor neurons. Motor neurons very strongly express GluR3 and 4, while the expression of GluR2 and GluR1 is moderate and low, respectively. Expression of GluR5 is also low in the motor neurons. However, expression of NR1 and the glutamate-binding subunit of an NMDA receptor complex is very strong. These findings indicate that the subunit composition of the AMPA-type receptors regulating motor neurons is different from that of the AMPA-type receptors in the spinal sensory neurons, and that there are at least two kinds of glutamergic systems which regulate motor neurons: via AMPA-type receptors and via NMDA receptors.

AMPA, KA and NMDA receptors are expressed in the rat DRG neurones.

The localization of AMPA receptor subunits (GluR1-4), a KA receptor subunit (GluR5) and NMDA receptor subunits (NR1 and NRgbs; the glutamate binding subunits of an NMDA receptor complex) was investigated using immunohistochemistry and in situ hybridization histochemistry in the rat dorsal root ganglion. Small neurones expressed GluR1-, GluR2/3-like immunoreactivities and GluR5, NR1, NRgbs mRNAs, while large neurones expressed GluR2/3-like immunoreactivity and NR1 and NRgbs mRNAs. These data suggest that the glutamatergic system plays an important role in the primary sensory afferent systems and that the composition of glutamate receptors differs according to the cell size.

Distribution of the substance P receptor (NK-1 receptor) in the central nervous system

The overall distribution of mRNA for the substance P receptor (NK-1) was investigated in the rat brain by in situ hybridization histochemistry using synthetic oligonucleotide probes. The NK-1 positive cells were distributed throughout the brain. Among the positive cells an intense hybridization signal was observed in the basal ganglia and the dorsal tegmental areas. In the hippocampus, hypothalamus, midbrain, and medulla oblongata, weak to moderate positive signal were recorded in various areas. Comparing our results with those of previous workers on the localization of substance P using both immunohistochemistry and in situ hybridization various relationships between the ligand and its receptor localization were characterized. Generally the areas containing the abundant substance P immunoreactive (SP-IR) terminals were associated with NK-1 mRNA positive cells, some examples of this can be seen in the medial amygdaloid nucleus, the locus coeruleus, and the dorsal raphe nucleus. Contrary to these matched areas, we could not detect NK-1 mRNA positive cells in the substantia nigra, where there is a strong projection from the caudate putamen containing the substance P. Similarly the substantia gelatinosa in the trigeminal tract nucleus and the dorsal horn of the spinal cord where numerous SP-IR fibers are located, did not show abundant NK-1 mRNA positive cells. Only a few weakly labeled positive cells could be found.