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Shinichi Kohsaka

Publications and source records attributed to Shinichi Kohsaka.

At least 19 recordsLinked to original sources
PubMed2006

The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release.

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca2+-dependent exocytosis, which was abolished by the Ca2+-channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A.

Animals↗
PubMed2006

Monoubiquitylation of GGA3 by hVPS18 regulates its ubiquitin-binding ability.

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins), constitute a family of monomeric adaptor proteins and are associated with protein trafficking from the trans-Golgi network to endosomes. Here, we show that GGA3 is monoubiquitylated by a RING-H2 type-ubiquitin ligase hVPS18 (human homologue of vacuolar protein sorting 18). By in vitro ubiquitylation assays, we have identified lysine 258 in the GAT domain as a major ubiquitylation site that resides adjacent to the ubiquitin-binding site. The ubiquitylation is abolished by a mutation in either the GAT domain or ubiquitin that disrupts the GAT-ubiquitin interaction, indicating that the ubiquitin binding is a prerequisite for the ubiquitylation. Furthermore, the GAT domain ubiquitylated by hVPS18 no longer binds to ubiquitin, indicating that ubiquitylation negatively regulates the ubiquitin-binding ability of the GAT domain. These results suggest that the ubiquitin binding and ubiquitylation of GGA3-GAT domain are mutually inseparable through a ubiquitin ligase activity of hVPS18.

ADP-Ribosylation Factors↗
PubMed2006

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding.

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.

Amino Acid Sequence↗
PubMed2006

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion.

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.

Animals↗
PubMed2006

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.

Animals↗
PubMed2006

Differential suppression of endotoxin-inducible inflammatory cytokines by nuclear factor kappa B (NFkappaB) inhibitor in rat microglia.

The molecular mechanism by which the deleterious cytokines interleukin 1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) are induced in endotoxin-stimulated microglia was investigated from the viewpoint of signal transduction. Neither cytokine is produced in nonstimulated rat microglia, but both are remarkably induced by stimulation with endotoxin lipopolysaccharide (LPS). LPS-inducible IL-1beta was significantly suppressed by pretreatment with the nuclear factor kappa B (NFkappaB) inhibitor ammonium pyrrolidine dithiocarbamate (APDC), but TNFalpha was not. APDC was actually confirmed to suppress the degradation of IkappaBalpha and IkappaBbeta in microglia, indicating a role for the inhibitor of NFkappaB activation. Taken together, these results suggest that the induction of IL-1beta and TNFalpha in endotoxin-stimulated microglia is differentially regulated at the level of NFkappaB activation.

Animals↗
PubMed2006

Autotaxin expression is enhanced in frontal cortex of Alzheimer-type dementia patients.

We searched for genes differentially expressed in the frontal cortices of Alzheimer-type dementia (ATD) patients compared with those of non-ATD controls using DNA microarray and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. Here we show that the expression level of the autotaxin (also called lysophospholipase D or ecto-nucleotide pyrophosphatase/phosphodiesterase 2) gene was significantly greater in ATD cortices than in non-ATD cortices. In both ATD and non-ATD groups, the expression levels were greater in patients with the apoE epsilon3/epsilon4 genotype than in patients with the apoE epsilon3/epsilon3 genotype, although the differences were not statistically significant. These observations suggest that expression of the autotaxin gene and cell signaling by lysophosphatidic acid may be involved in the pathology of ATD, and that this cell signaling pathway may be a potential target of treatments for ATD.

Aged↗
PubMed2006

Nonparticipation of nuclear factor kappa B (NFkappaB) in the signaling cascade of c-Jun N-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFalpha) induction in lipopolysaccharide (LPS)-stimulated microglia.

The molecular mechanism of cytotoxic cytokine tumor necrosis factor alpha (TNFalpha) induction in microglia remains to be clarified. We have previously reported that p38 mitogen-activated protein kinase (p38MAPK) is an important signaling molecule for the induction of TNFalpha in lipopolysaccharide (LPS)-stimulated microglia. Recently, we have shown that c-Jun N-terminal kinase (JNK) is associated with the induction of TNFalpha. Furthermore, using an NFkappaB inhibitor (SN50), we discovered that activation of nuclear factor kappaB (NFkappaB) may also be linked to TNFalpha induction. We therefore examined the relationship between NFkappaB and the two MAPKs (p38MAPK and JNK) in the signaling cascade of TNFalpha induction in LPS-stimulated microglia. NFkappaB inhibitor SN50 decreased the induction of TNFalpha under the suppressed NFkappaB activation. However, SN50 was found to prevent the activation of MKK3/6-p38MAPK and MKK4-JNK pathways. On the other hand, the other NFkappaB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) neither prevented the activation of p38MAPK and JNK nor inhibited TNFalpha induction in LPS-stimulated microglia, although it was confirmed to serve as an NFkappaB inhibitor. These results suggest that both MKK3/6-p38MAPK and MKK4-JNK pathways are important signaling cascades leading to the induction of TNFalpha in LPS-stimulated microglia, but that NFkappaB itself is not required for this induction.

Animals↗
PubMed2006

Fyn is required for haloperidol-induced catalepsy in mice.

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.

Animals↗
PubMed2006

Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role.

Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X(7) receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X(7) receptor-mediated TNF release, whilst suppressing LPS-induced TNF release but without affecting TNF mRNA expression via activation of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca(2+) levels, which was abolished by specific blockers of alpha7 nAChRs. However, this response was independent of extracellular Ca(2+) and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP(3) receptor. Repeated experiments showed that currents were not detected in nicotine-stimulated microglia. Moreover, nicotine modulation of LPS-induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post-transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca(2+) response in P2X(7) receptor-activated microglia. In conclusion, microglial alpha7 nAChRs might drive a signaling process involving the activation of PLC and Ca(2+) release from intracellular Ca(2+) stores, rather than function as conventional ion channels. This novel alpha7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.

Animals↗
PubMed2005

Ubiquitylation and degradation of serum-inducible kinase by hVPS18, a RING-H2 type ubiquitin ligase.

Serum-inducible kinase (SNK) is a member of polo-like kinases that serve as regulators of multiple events during cell division. Rapid changes in the activity and abundance of SNK were reported after the serum stimulation and after the activation of synaptic transmission in the brain. Yet the detailed mechanisms that control the level of SNK protein have not been fully elucidated. In this report, we show that the RING-H2 domain of hVPS18 (human vacuolar protein sorting 18) has a genuine ubiquitin ligase (E3) activity. Using the yeast two-hybrid screening, we identify SNK as a candidate substrate of hVPS18. The half-life of SNK is increased in HeLa cells that down-regulated hVPS18 by lentivirus-mediated small hairpin RNA interference. Furthermore, the delayed entry into S phase is observed in HeLa cells overexpressing hVPS18. These results suggest that hVPS18 may play an important role in regulation of SNK activity through its ubiquitin ligase.

Animals↗
PubMed2005

Axonal injury in experimental herpes simplex encephalitis.

Using beta-amyloid precursor protein immunolabeling, we have detected axonal injury in experimental herpes simplex encephalitis. beta-amyloid precursor protein-specific signals were found in the mouse brain as either puncta or axon-like structures. They appeared where infected neurons were undergoing apoptosis and Iba1-immunopositive microglia transformed themselves into macrophages. These results show, for the first time, that axonal injury, i.e., functional disturbance of the fast axonal transport, can take place during the course of acute viral encephalitis.

Amyloid beta-Protein Precursor↗
PubMed2005

Receptor related to tyrosine kinase RYK regulates cell migration during cortical development.

Mammalian RYK is a receptor related to tyrosine kinase without detectable catalytic activity. We have previously reported that rat RYK is dominantly expressed in neural progenitor cells and mature neurons in the developing central nervous system. Mouse RYK has been found to bind to EphB2/B3 receptors, which have diverse functions during development. In this study, we demonstrated that RYK, EphB2, EphB3, ephrinB1, and ephrinB2 are expressed in embryonic brain. In vitro analysis using COS-7 cells revealed binding between rat RYK and EphB3, and that the RYK deletion mutant without extracellular leucine-rich motifs lacked this binding ability. To investigate the function of RYK in vivo, embryonic cortical slice cultures were analyzed after electroporation of expression plasmids for RYK or its deletion mutants. The results showed that overexpression of RYK suppressed cell migration from the ventricular zone to the pial surface, however, overexpression of the RYK deletion mutant without leucine-rich motifs had no effect on cell migration. These results suggest that RYK regulates cell migration during mammalian cortical development through the binding to Eph receptors.

Animals↗
PubMed2005

The Pax6 isoform bearing an alternative spliced exon promotes the development of the neural retinal structure.

The vertebrate retina has an area where visual cells are closely packed for proper vision that is known as a fovea, an area centralis or a visual streak. The molecular mechanism that regulates the formation of these structures and visual cell gradients is unknown. The transcription factor Pax6 is a master regulator of eye development. A Pax6 isoform that contains an exon 5a-encoded 14 amino acid insertion in its paired domain, Pax6(+5a), has different DNA-binding properties compared with the Pax6(-5a) isoform. Little is known about the functional significance of Pax6(+5a). Here, we show that Pax6(+5a) is expressed especially in the retinal portion where visual cells accumulate during eye development and, when overexpressed, induces a remarkable well-differentiated retina-like structure. Pax6(+5a) proteins that bear point mutations that are found in patients with foveal hypoplasia are unable to induce these ectopic retina-like structures. We propose that Pax6(+5a) induces a developmental cascade in the prospective fovea, area centralis or visual streak region that leads to the formation of a retinal architecture bearing densely packed visual cells.

Alternative Splicing↗
PubMed2005

Axotomy-dependent urokinase induction in the rat facial nucleus: possible stimulation of microglia by neurons.

The phenomenon in which urokinase-type plasminogen activator (uPA) is induced in the axotomized facial nucleus suggests an interaction between injured motoneurons and microglia. We examined the relation of neurons and microglia to the induction of uPA in vitro. The amount of uPA released from a co-culture of neurons and microglia was much greater than the addition of that from each alone, suggesting the occurrence of an interaction between the two. The analysis of conditioned neuronal medium (CNM)-effects on microglia and conditioned microglial medium (CMM)-effects on neurons revealed that microglia enhance uPA release in response to CNM, rather than vice versa. Characterization of the CNM-effect on microglia demonstrated that CNM enhances not only uPA release but also the specific activity of acid phosphatase and 5'-nucleotidase in microglia. The profile of microglial activation caused by CNM was quite different from that caused by lipopolysaccharide (LPS)-activation. These results suggest that a specific soluble constituent(s) derived from neurons activates microglia by a mechanism different from LPS. As a candidate molecule for the microglial activation, brain-derived neurotrophic factor was detected in the CNM. Thus, uPA induction in the axotomized facial nucleus may be explained by a neuronal stimulus leading to uPA induction in microglia.

Acrylic Resins↗
PubMed2004

Enhancement of urokinase-type plasminogen activator (uPA) secretion, but not that of substrate plasminogen (PGn), by rat microglia stimulated with neuronal conditioned medium.

Assuming the presence of intercellular interactions between injured motoneurons and microglia in the axotomized facial nucleus, we investigated the effects of neuronal conditioned medium (NCM) on the release of urokinase-type plasminogen activator (uPA) from microglia. Zymography revealed that NCM markedly enhanced the release of uPA from microglia, although NCM itself did not contain a significant amount of uPA. In contrast, the secretion of plasminogen (PGn), a substrate of uPA, was not promoted by the NCM treatment. The specific effect of NCM was found to be quite distinct from that of microglial activators, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which reduce uPA release from microglia. In summary, Neuron-derived soluble molecules appear to stimulate microglia to enhance the production/release of uPA, but not that of PGn, by a mechanism independent of the activation reaction by IFN-gamma or LPS.

Animals↗
PubMed2004

The upregulated expression of sonic hedgehog in motor neurons after rat facial nerve axotomy.

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that of beta-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.

Adenoviridae↗