[Show abstract][Hide abstract] ABSTRACT: We have shown that two multidomain adaptor proteins, p140Cap and vinexin, interact with each other and are likely to be involved in neurotransmitter release. Because the basic molecular mechanism governing neurotransmitter and insulin secretion is conserved, these two proteins may also to play pivotal roles in insulin secretion. We therefore performed some characterization of p140Cap and vinexin in pancreas of a wild-type rat or a spontaneous type 2 diabetes mellitus (DM) model, the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. These two proteins were detected in Wistar rat pancreas by Western blotting. Immunohistochemistry revealed that p140Cap and vinexin are enriched in β and α cells, respectively, in the rat pancreas. We then found that pancreatic islet structure was disorganized in the OLETF rat with hyperinsulinemia or with hyperglycemia, based on immunohistochemical analyses of vinexin. In β cells of these model rats, p140Cap was distributed in a cytoplasmic granular pattern as in the control rats, although its expression was reduced to various extents from cell to cell. These results may suggest possible involvement of p140Cap in insulin secretion, and reduction of p140Cap might be related to abnormal insulin secretion in DM.
Medical Molecular Morphology 01/2013; · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.
Medical Molecular Morphology 12/2012; 45(1):22-8. · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The membrane-associated guanylate kinase with inverted organization (MAGI) proteins consist of three members, MAGI-1, MAGI-2 (also known as S-SCAM), and MAGI-3. Although MAGI-2 has been analyzed and shown to interact with a variety of postsynaptic proteins, functional analyses and characterization of MAGI-1 in neuronal tissues have been rare. In this study, we prepared a specific antibody against MAGI-1, anti-MAGI-1, and carried out biochemical and morphological analyses of MAGI-1 in rat neuronal tissues. By Western blotting, a high level of MAGI-1 was detected in nervous tissues, especially in olfactory bulb. Biochemical fractionation clarified that MAGI-1 was relatively enriched in the synaptosomal vesicle and synaptic plasma membrane fractions, whereas MAGI-2 and MAGI-3 appeared to be in the synaptic plasma membrane and postsynaptic density fractions. Immunofluorescent analyses revealed diffuse distribution of MAGI-1 in the cell body and processes of primary cultured rat hippocampal neurons, whereas MAGI-2 and MAGI-3 were likely to be enriched at synapses. Immunohistochemical analyses demonstrated that MAGI-1 was expressed in Purkinje cells, in hypocampal neurons in CA1 region, in the glomerulus region of olfactory bulb, and at the dorsal root entry zone in embryonic rat spinal cord. These results suggest neuronal roles of MAGI-1 different from those of MAGI-2/3.
Journal of Neuroscience Research 05/2012; 90(9):1776-81. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In adult rat brains, brain-derived neurotrophic factor (BDNF) rhythmically oscillates according to the light-dark cycle and exhibits unique functions in particular brain regions. However, little is known of this subject in juvenile rats. Here, we examined diurnal variation in BDNF and neurotrophin-3 (NT-3) levels in 14-day-old rats. BDNF levels were high in the dark phase and low in the light phase in a majority of brain regions. In contrast, NT-3 levels demonstrated an inverse phase relationship that was limited to the cerebral neocortex, including the visual cortex, and was most prominent on postnatal day 14. An 8-h phase advance of the light-dark cycle and sleep deprivation induced an increase in BDNF levels and a decrease in NT-3 levels in the neocortex, and the former treatment reduced synaptophysin expression and the numbers of synaptophysin-positive presynaptic terminals in cortical layer IV and caused abnormal BDNF and NT-3 rhythms 1 week after treatment. A similar reduction of synaptophysin expression was observed in the cortices of Bdnf gene-deficient mice and Ca(2+)-dependent activator protein for secretion 2 gene-deficient mice with abnormal free-running rhythm and autistic-like phenotypes. In the latter mice, no diurnal variation in BDNF levels was observed. These results indicate that regular rhythms of BDNF and NT-3 are essential for correct cortical network formation in juvenile rodents.
Journal of Biological Chemistry 06/2011; 286(24):21478-87. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.
[Show abstract][Hide abstract] ABSTRACT: Septins are a family of conserved guanosine triphosphate/guanosine diphosphate-binding proteins implicated in a variety of cellular functions such as cell cycle control and cytokinesis. Although several members of septin family, including Septin 14 (Sept14), are abundantly expressed in nervous tissues, little is known about their physiological functions, especially in neuronal development. Here, we report that Sept14 is strongly expressed in the cortical plate of developing cerebral cortex. Knockdown experiments by using the method of in utero electroporation showed that reduction of Sept14 caused inhibition of cortical neuronal migration. Whereas cDNA encoding RNA interference-resistant Sept14 rescued the migration defect, the C-terminal deletion mutant of Sept14 did not. Biochemical analyses revealed that C-terminal coiled-coil region of Sept14 interacts with Septin 4 (Sept4). Knockdown experiments showed that Sept4 is also involved in cortical neuronal migration in vivo. In addition, knockdown of Sept14 or Sept4 inhibited leading process formation in migrating cortical neurons. These results suggest that Sept14 is involved in neuronal migration in cerebral cortex via interaction with Sept4.
Molecular biology of the cell 02/2010; 21(8):1324-34. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Septins, a conserved family of GTP/GDP-binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post-natal development. Morphological analyses revealed that Sept8 is enriched at pre-synapses. Using yeast two-hybrid screening, we identified vesicle-associated membrane protein 2 (VAMP2), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome-associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by synaptosome-associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.
Journal of Neurochemistry 03/2009; 108(4):867-80. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.
Journal of Neurochemistry 09/2008; 107(1):61-72. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circadian variation in the expression of brain-derived neurotrophic factor (BDNF) indicates that BDNF is involved in the regulation of diurnal rhythms in a variety of biological processes. However, it is still unclear which brain regions alter their BDNF levels in response to external light input. Therefore, in selected brain regions of adult male rats, we investigated diurnal variation, as well as the effects of a single eight-hour phase advance of the light-dark cycle, on the levels of BDNF and of other neurotrophins. The cerebellum, hippocampus and cerebral cortex containing visual cortex (VCX) showed diurnal variation in BDNF protein levels and the VCX also in NT-3 levels. In the VCX and the region containing the entorhinal cortex and amygdala (ECX), BDNF protein levels were increased 12 h after the phase advance, while BDNF mRNA levels were increased significantly in the VCX and slightly in the ECX after 4 h. After one week, however, BDNF protein levels were reduced in eight brain regions out of 13 examined. BDNF levels in the ECX and VCX were significantly different between light rearing and dark rearing, while a hypothyroid status did not produce an effect. Cyclic AMP responsive element-binding protein (CREB), a transcription factor for BDNF, was greatly activated by the phase advance in the ECX and VCX, suggesting the existence of CREB-mediated pathways of BDNF synthesis that are responsive to external light input.
Journal of Neurochemistry 09/2008; 106(5):2131-42. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SEPT9 is a member of the cytoskeleton-related septin family, which is highly expressed in glia cells in neuronal tissues. Sequence alterations in SEPT9 are known to cause hereditary neuralgic amyotrophy (HNA) but precise cellular consequences have yet to be determined. Since SEPT9 is thought to function through interaction with other septins and small GTPase Rho-mediated signaling, we analyzed the properties of HNA-associated SEPT9 missense variants, SEPT9F (c.278C>T/p.Ser93Phe in SEPT9_v3; NM_006640.3) and SEPT9W (c.262C>T/p.Arg88Trp in SEPT9_v3). We found both sequence variants, but not the wild type, to form filaments with SEPT4 along stress fibers in mesenchymal mouse mammary gland NMuMG cells. In the epithelial cells, the variants, but not the wild type, were colocalized with SEPT11 at cell-cell junctions. In addition, although septin filaments containing SEPT9_v3 were disrupted by Rho/Rhotekin signaling, this was not the case with SEPT9F and SEPT9W. Sequence variations in SEPT9 causing HNA are thus likely to alter modes of interaction with partner molecules in cells, and consequently contribute to the pathogenesis of HNA.
Human Mutation 11/2007; 28(10):1005-13. · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinalpha, beta and gamma, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage-dependent manner, and that vinexinalpha is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinalpha in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinbeta is reported to be phosphorylated by extracellular signal-regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinalpha. We thus constructed a site- and phosphorylation state-specific antibody to monitor the ERK-mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co-localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin-dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.
Journal of Neurochemistry 02/2007; 100(2):545-54. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rhotekin, an effector of Rho, is highly expressed in the brain but its function(s) in neurons is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin by yeast two-hybrid screening of human brain cDNA library. We then found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in COS7 cells and the rat brain. The C-terminal three amino acids of Rhotekin were essential for the interaction with Lin-7B. Their binding affinity became increased in the presence of active RhoA in the COS7 cell expression system. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.
Neuroscience Research 01/2007; 56(4):347-55. · 2.15 Impact Factor