Hiroyuki Sakagami

Rac1 is required for the formation of three germ layers during gastrulation

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.

Localization of mRNAs of voltage-dependent Ca2+-channels : four subtypes of α1- and β-subunits in developing and mature rat brain

Abstract The heterogeneous gene expression for four subtypes of al (A,B,C,D)- and β (β1,β2,β3,β4)-subunits of voltage-dependent calcium channels was demonstrated in developing and adult rat brain by in situ hybridization histochemistry. In the adult rat brain the gene expression for A- and B-subtypes was predominant in the cerebellar cortex and hippocampal neuronal layers, with the A-subtype expressed most intensely in the Purkinje cells, while the expression for C- and D-subtypes was predominant in the olfactory mitral and granule cells and the dentate granule cells. The expression of β1-mRNA was prominent in the olfactory mitral cells and dentate granule cells whereas that of β2-mRNA was evident in the hippocampal neuronal layers and cerebellar Purkinje cells. The expression of β3-mRNA was prominent in the olfactory mitral and internal granule cells and medial habenula, whereas that of β4-mRNA in the olfactory mitral cells and cerebellar Purkinje and granule cells. Comparison between the expression patterns for individual α- and β-subunits suggests that the β4-subunit contributes to P-type channel, whereas the β1- and β3-subunits contribute respectively to D- and C-subtypes of L-type channels, although dissociation in the expression patterns were also noted in several brain regions. In addition to neuronal populations, the gene expression for the C-subtype of L-type channel was detected at substantial level in glial cells. In developing brains, the genes for the all subtypes of α1- and β-subunits were expressed in the mantle zones, but not the ventricular zones, of the entire neuraxis and the expression was more or less attenuated during early postnatal periods in most of the brain regions except for the olfactory bulb. hippocampas and cerebellar cortex, suggesting that the Ca 2+-channels are intimately involved in the neuronal differentiation.

ERK5 activation of MEF2-mediated gene expression plays a critical role in BDNF-promoted survival of developing but not mature cortical neurons

Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase family whose biological function in the CNS has not been defined. In contrast to ERK1 and ERK2, which are activated by neurotrophins (NTs), cAMP, and neuronal activity in cortical neurons, ERK5 is activated only by NTs. Here, we report that ERK5 expression is high in the brain during early embryonic development but declines as the brain matures to almost undetectable levels by postnatal day (P) 49. Interestingly, expression of a dominant-negative ERK5 blocked brain-derived neurotrophic factor protection against trophic withdrawal in primary cortical neurons cultured from embryonic day (E) 17 but not P0. Furthermore, expression of a dominant-negative ERK5 induced apoptosis in E17 but not P0 cortical neurons maintained in the presence of serum. We also present evidence that ERK5 protection of E17 cortical neurons may be mediated through myocyte enhancer factor 2-induced gene expression. These data suggest that ERK5 activation of myocyte enhancer factor 2-induced gene expression may play an important and novel role in the development of the CNS by mediating NT-promoted survival of embryonic neurons.

Altered emotional behavioral responses in mice lacking brain-type fatty acid-binding protein gene

Brain‐type fatty acid‐binding protein (B‐FABP) belongs to a family of intracellular lipid‐binding proteins. B‐FABP exhibits a binding affinity to long‐chain fatty acids (FAs) whose effects on brain functions including development, emotion, learning and memory have been proposed. B‐FABP is localized in the ventricular germinal cells in embryonic brain and astrocytes in developing and mature brain of rodents. In the present study we generated the mouse harboring a null mutation in the B‐FABP gene and studied its phenotype. B‐FABP mutant mice exhibited the enhanced anxiety and increased fear memory as well as the decreased content of docosahexaenoic acid (DHA) in their brain during the neonatal period without detection of any histological changes in the brain. In the adult brain, B‐FABP was localized more numerously to the astrocytes in the amygdala and septal area than to those in the hippocampal area. Analysis of FA content in the amygdala of adult brain revealed that arachidonic and palmitic acids increased significantly in the mutant mice compared with wild‐type. Furthermore, the response of N‐methyl‐d‐aspartate receptor‐mediated current to DHA in isolated neurons from B‐FABP mutant brain was significantly decreased compared with that of wild‐type, while no significant differences were detected in behavioral responses related to the spatial learning/memory or in the hippocampal long‐term potentiation. These data indicate that B‐FABP is crucially involved in the fear memory and anxiety through its binding with FAs and/or its own direct effects on pertinent metabolism/signaling of FAs.

Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2)

The rabbit polyclonal antibody against rat organic anion transporting polypeptide 2 (oatp2) was raised and immunoaffinity‐purified. Western blot analysis for oatp2 detected two bands (∼74 and 76 kDa) in rat brain and a single band (76 kDa) in the liver. By immunohistochemical analysis, the oatp2 immunoreactivity was specifically high at the basolateral membrane of rat hepatocytes. Functionally, the oatp2‐expressing oocytes were found to transport dehydroepiandrosterone sulfate, δ1 opioid receptor agonist [d‐Pen2,d‐Pen5]enkephalin, Leu‐enkephalin, and biotin significantly, as well as the substrates previously reported. These data reveal the exact distribution of the rat oatp2 at the protein level in the liver, and that oatp2 appears to be involved in the multispecificity of the uptaking substrates in the liver and brain.

COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12-CXCR4 chemokine systems

Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)-2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3-/- mice and EP4-/- mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins.

Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse.

Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

Kinase-Dead Knock-In Mouse Reveals an Essential Role of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Dendritic Spine Enlargement, Long-Term Potentiation, and Learning

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIα, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIα, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIα knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIα (K42R) mice, kinase activity was reduced to the same level as in CaMKIIα-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral–CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIα was preserved. In addition, CaMKIIα (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIα is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.

Importin α transports CaMKIV to the nucleus without utilizing importin β

Ca2+/calmodulin‐dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element‐binding protein‐mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin α is able to carry CaMKIV into the nucleus without the need for importin β or any other soluble proteins in digitonin‐permeabilized cells. An importin β binding‐deficient mutant (ΔIBB) of importin α also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin β‐independent manner. While CaMKIV directly interacted with the C‐terminal region of importin α, the CaMKIV/importin α complex did not form a ternary complex with importin β, which explains the nonrequirement of importin β for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin α‐ΔIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin α solely carries a cargo protein into the nucleus without utilizing the classical importin β‐dependent transport system.

Cloning and nucleotide sequence of cDNA encoding Asp-hemolysin from Aspergillus fumigatus

The nucleotide sequence of a cDNA encoding Asp-hemolysin from Aspergillus fumigatus was clarified. The deduced amino acid sequence was shown to contain a set of negatively charged domains similar to portions of the cysteine-rich sequence in the ligand-binding domain of LDL-receptor. A potential signal sequence was also identified in the N-terminal domain of the deduced amino acid sequence.