Tatsuo Furuyama

Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues.

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

Forkhead transcription factor FOXO1 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation

A forkhead-type transcription factor, DAF-16, is located in the most downstream part of the insulin signalling pathway via PI3K (phosphoinositide 3-kinase). It is essential for the extension of life-span and is also involved in dauer formation induced by food deprivation in Caenorhabditis elegans. In the present study, we addressed whether or not FOXO members AFX, FKHR (forkhead homologue in rhabdomyosarcoma) and FKHRL1 (FKHR-like protein 1), mammalian counterparts of DAF-16, are involved in starvation stress. We found a remarkable selective induction of FKHR and FKHRL1 transcripts in skeletal muscle of mice during starvation. The induction of FKHR gene expression was observed at 6 h after food deprivation, peaked at 12 h, and returned to the basal level by 24 h of refeeding. The induction was also found in skeletal muscle of mice with glucocorticoid treatment. Moreover, we found that the levels of PDK4 (pyruvate dehydrogenase kinase 4) gene expression were up-regulated through the direct binding of FKHR to the promoter region of the gene in C2C12 cells. These results suggest that FKHR has an important role in the regulation of energy metabolism, at least in part, through the up-regulation of PDK4 gene expression in skeletal muscle during starvation.

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.

localizations of α1 and β1 subunits of soluble guanylate cyclase in the rat brain

Abstract We studied the localizations of α1 and β1 subunits of soluble guanylate cyclase using in situ hybridization. The β subunit was widely distributed in most neurons throughout the brain, with different levels of expression. The α1 subunit was also distributed throughout the brain; however, it was located in more limited regions. Both subunits were expressed markedly in the glomerular layer of the olfactory bulb, dorsal and ventral striatum, and several regions in the brainstem. Regions with little or no α1 subunit expression, but with marked expression of the β1 subunit included the olfactory bulb except for the glomerular layer, pyramidal cell layer in CA1 and granular cell layer in the dentate gyrus of the hippocampus, and many brainstem nuclei. The above regions expressing both subunits are suggested to contain active soluble guanylate cyclase as a target for nitric oxide, and thus may be involved in cellular signal transduction.

Identification of a Novel Transmembrane Semaphorin Expressed on Lymphocytes

Semaphorin (also known as collapsin) members are thought to be involved in axon guidance during neural network formation. Here, we report the isolation of a novel member, mouse semaphorin G (M-sema G), which encodes a semaphorin domain followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic domain. M-sema G is most closely related to M-sema F, which we previously reported, and semB and semC. These four members appear to constitute a transmembrane type subfamily in mouse semaphorins. In contrast to the predominant expression of M-sema F mRNAs in the nervous tissues, M-sema G mRNAs are strongly expressed in lymphoid tissues, especially in the thymus, as well as in the nervous tissues. The mRNAs are also detected in various cell lines from hematopoietic cells. By generating specific antibodies, we confirmed the strong expression of M-Sema G proteins on the surface of lymphocytes. These results provide the first evidence that semaphorin is expressed on lymphocytes and suggest that semaphorins may play an important role in the immune system, as well as in the nervous system.

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.

A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue.

Abstract: Mutations in the presenilin‐1 (PS‐1) and presenilin‐2 (PS‐2) genes account for the majority of cases of early‐onset familial Alzheimers disease (AD). Alternative splicing forms of the PS‐1 and PS‐2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS‐2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age‐matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia‐mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N‐acetylcysteine and diphenyl iodonium, suggesting that hypoxia‐mediated oxidant stress might, at least in part, underlie the alternative splicing of PS‐2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N‐terminal part of PS‐2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.

Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors

The Rho family GTPase has been implicated in plexin-B1, a receptor for Semaphorin 4D (Sema4D), mediating signal transduction. Rho may also play a function in this signaling pathway as well as Rac, but the mechanisms for Rho regulation are poorly understood. In this study, we have identified two kinds of PDZ domain-containing Rho-specific guanine nucleotide exchange factors (RhoGEFs) as proteins interacting with plexin-B1 cytoplasmic domain. These PDZ domain-containing RhoGEFs showed significant homology to human KIAA0380 (PDZ-RhoGEF) and LARG (KIAA0382), respectively. Both KIAA0380 and LARG could bind plexin-B1 and a deletion mutant analysis of plexin-B1, KIAA0380 and LARG revealed that KIAA0380 and LARG bound plexin-B1 cytoplasmic tail through their PDZ domains. The tissue distribution analysis indicated that plexin-B1 was co-localized with KIAA0380 and LARG in various tissues. Immunocytochemical analysis showed that LARG was recruited to plasma membrane by plexin-B1. These results suggest that PDZ domain-containing RhoGEFs play a role in Sema4D-plexin-B1 mediating signal transduction.

Sema4D stimulates axonal outgrowth of embryonic DRG sensory neurones

Several semaphorins are thought to function as potent inhibitors of axonal growth. We have found that Sema4D stimulates axonal outgrowth of embryonic dorsal root ganglion (DRG) neurones in stead of retraction. Neutralizing antibodies to Sema4D inhibit this action. This action appears to differ slightly from that on PC12 cells, because DRG neurones respond to Sema4D without addition of nerve growth factor (NGF), while PC12 cells do not. On the other hand, it is blocked by deprivation of endogenous NGF with antibodies to NGF and also by Trk‐inhibitor K252a, suggesting that endogenously produced‐NGF and the activation of Trk receptor are required for Sema4D‐action on DRG neurones. These indicate that neurite‐outgrowth promoting actions of Sema4D are similar between DRG neurones and PC12 cells, since NGF‐Trk signalling are required for these actions. Since Schwann cells can produce NGF, the contamination of these cells in our DRG culture might explain this action. In addition to plexin‐B1 that is known as a Sema4D receptor, binding experiments indicate plexin‐B2 as another receptor candidate for Sema4D. These plexins and Sema4D are expressed in embryonic DRGs. We suggest a new function of Sema4D as a neurite‐outgrowth stimulating, autocrine/paracrine factor in embryonic sensory neurones.

Identification of a member of mouse semaphorin family

Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M-Sema F. The N-terminal encodes a semaphorin domain that is similar between Sema I-III [6] followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a proline-rich intracellular domain. M-Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M-Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M‐Sema F. The N‐terminal encodes a semaphorin domain that is similar between Sema I–III [6] followed by a single putative immunoglobulin‐like domain, a transmembrane domain, and a proline‐rich intracellular domain. M‐Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M‐Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.