Biomedical subjects

Shigeo Okabe

Publications and source records attributed to Shigeo Okabe.

At least 19 recordsLinked to original sources

PubMed2007

Direct astrocytic contacts regulate local maturation of dendritic spines.

Astrocytes contribute on both development and function of synapses, but it remains unclear whether direct astrocytic contacts regulate development of individual synapses. Two-photon time-lapse imaging of astrocytic and dendritic protrusive activity revealed the correlation of astrocytic contacts with both lifetime and morphological maturation of dendritic protrusions. Astrocytic motility was essential in maturation of spines, because its suppression by manipulating Rac1-dependent signaling in astrocytes resulted in induction of longer, filopodia-like dendritic protrusions. Manipulation of ephrin/Eph-dependent neuron-astrocyte signaling suggested involvement of this signaling pathway in astrocyte-dependent stabilization of newly generated dendritic protrusions. Our data support a model in which astrocytic protrusive activity in development acts as a key local regulator for stabilization of individual dendritic protrusions and subsequent maturation into spines.

Animals↗

PubMed2006

Myosin-Va facilitates the accumulation of mRNA/protein complex in dendritic spines.

mRNA localization has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion, and allowing the local control of protein synthesis in neurons. In neuronal dendrites, the localization and translocation of mRNA is considered as one of the molecular bases of synaptic plasticity. Recent imaging and functional studies revealed that several RNA-binding proteins form a large messenger ribonucleoprotein (mRNP) complex that is involved in transport and translation of mRNA in dendrites. However, the mechanism of mRNA translocation into dendritic spines is unknown. Here, we show that an actin-based motor, myosin-Va, plays a significant role in mRNP transport in neuronal dendrites and spines. Myosin-Va was Ca2+-dependently associated with TLS, an RNA-binding protein, and its target RNA Nd1-L, an actin stabilizer. A dominant-negative mutant or RNAi of myosin-Va in neurons suppressed TLS accumulation in spines and further impaired TLS dynamics upon activation of mGluRs. The TLS translocation into spines was impeded also in neurons prepared from myosin-Va-null dilute-lethal (dl) mice, which exhibit neurological defects. Our results demonstrate that myosin-Va facilitates the transport of TLS-containing mRNP complexes in spines and may function in synaptic plasticity through Ca2+ signaling.

Animals↗

PubMed2006

Differential control of postsynaptic density scaffolds via actin-dependent and -independent mechanisms.

Organization and dynamic remodeling of postsynaptic density (PSD) are thought to be critical in postsynaptic signal transduction, but the underlying molecular mechanisms are not well understood. We show here that four major scaffolding molecules, PSD-95, GKAP, Shank, and PSD-Zip45, show distinct instability in total molecular content per synapse. Fluorescence recovery after photobleaching also confirmed their distinct turnover rates. Among the PSD molecules examined, PSD-95 was most stable, but its elimination did not influence the dynamics of its direct binding partner GKAP. Multiple interactions of scaffolding molecules with the actin cytoskeleton have suggested their importance in both maintenance and remodeling of the PSD. Indeed, acute pharmacological disruption of F-actin rapidly eliminated the dynamic fraction of GKAP, Shank, and PSD-Zip45, without changing synaptic localization of PSD-95. GKAP content in synapses increased after pharmacological enhancement of neuronal activity, whereas Shank and PSD-Zip45 content showed reduction. Inhibition of F-actin dynamics prevented activity-dependent redistribution of all three scaffolds. We also assessed involvement of glutamate receptors in the regulation of PSD dynamics. Genetic manipulations eliminating either NMDA receptors or metabotropic glutamate receptors did not primarily influence mobility of their binding scaffolds. These results collectively indicate a critical role of filamentous actin in determining the extent of dynamic reorganization in PSD molecular composition.

Actins↗

PubMed2006

Antipsychotic medication and cognitive function in schizophrenia.

Antipsychotic polypharmacy and excessive dosing still prevail worldwide in the treatment of schizophrenia, while their possible association with cognitive function has not well been examined. We examined whether the "non-standard" use of antipsychotics (defined as antipsychotic polypharmacy or dosage >1,000 mg/day of chlorpromazine equivalents) is associated with cognitive function. Furthermore, we compared cognitive function between patients taking only atypical antipsychotics and those taking only conventionals. Neurocognitive functions were assessed in 67 patients with chronic schizophrenia and 92 controls using the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale-Revised (WAIS-R), the Wisconsin Card Sorting Test (WCST), and the Advanced Trail Making Test (ATMT). Patients showed markedly poorer performance than controls on all these tests. Patients on non-standard antipsychotic medication demonstrated poorer performance than those on standard medication on visual memory, delayed recall, performance IQ, and executive function. Patients taking atypical antipsychotics showed better performance than those taking conventionals on visual memory, delayed recall, and executive function. Clinical characteristics such as duration of medication, number of hospitalizations, and concomitant antiparkinsonian drugs were different between the treatment groups (both dichotomies of standard/non-standard and conventional/atypical). These results provide evidence for an association between antipsychotic medication and cognitive function. This association between antipsychotic medication and cognitive function may be due to differential illness severity (e.g., non-standard treatment for severely ill patients who have severe cognitive impairment). Alternatively, poorer cognitive function may be due in part to polypharmacy or excessive dosing. Further investigations are required to draw any conclusions.

Adult↗

PubMed2006

Telencephalin slows spine maturation.

Dendritic filopodia are highly dynamic structures, and morphological maturation from dendritic filopodia to spines is intimately associated with the stabilization and strengthening of synapses during development. Here, we report that telencephalin (TLCN), a cell adhesion molecule belonging to the Ig superfamily, is a negative regulator of spine maturation. Using cultured hippocampal neurons, we examined detailed localization and functions of TLCN in spine development and synaptogenesis. At early stages of synaptogenesis, TLCN immunoreactivity gradually increased and was present in dendritic shafts and filopodia. At later stages, TLCN tended to be excluded from mature spine synapses in which PSD-95 (postsynaptic density-95) clusters were apposed to presynaptic synaptophysin clusters. To elucidate the function of TLCN in spine maturation, we analyzed the dendrite morphology of TLCN-overexpressing and TLCN-deficient neurons. Overexpression of TLCN caused a dramatic increase in the density of dendritic filopodia and a concomitant decrease in the density of spines. Conversely, TLCN-deficient mice showed a decreased density of filopodia and an acceleration of spine maturation in vitro as well as in vivo. These results demonstrate that TLCN normally slows spine maturation by promoting the filopodia formation and negatively regulating the filopodia-to-spine transition. In addition, we found that spine heads of mature neurons were wider in TLCN-deficient mice compared with wild-type mice. Thus, the preservation of immature synapses by TLCN may be an essential step for refinement of functional neural circuits in the telencephalon, that take charge of higher brain functions such as learning, memory, and emotion.

Animals↗

PubMed2005

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology.

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.

Actins↗

PubMed2005

Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3-CA1 synapses in hippocampal slice cultures.

Dendritic spines are highly motile structures, but the extent and mode of coordination in motility between spines and presynaptic varicosities with synaptic contacts is not clear. To analyze movements of dendritic spines and axonal varicosities simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. Varicosities and spines were visualized using two-photon microscopy to detect close association of two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall structural changes between varicosities and spines was correlated, while the direction of short-term volume changes was regulated independently. Furthermore, alterations of dendritic morphology induced by strong electrical stimulation had little effects on their association. These results indicate the presence of local regulatory mechanisms to coordinate presynaptic and postsynaptic motility.

Animals↗

PubMed2005

Direct visualization of cell movement in the embryonic olfactory bulb using green fluorescent protein transgenic mice: evidence for rapid tangential migration of neural cell precursors.

We analyzed motile behavior of neuronal precursor cells in the intact olfactory bulbs (OBs) using transgenic mice expressing GFP under the control of T alpha 1 tubulin promoter. In the olfactory bulbs at the embryonic days 12.5-14.5, a large number of immature neurons expressed GFP in this transgenic line. Embryonic OBs were maintained in an organ culture system and the migratory behavior of GFP-positive cells was analyzed by time-lapse confocal microscopy. We observed rapid tangential movement of GFP-positive cells in the ventral olfactory bulb. In contrast to the typical bipolar morphology of translocating immature neurons within the developing cortex, the motile cells had neither leading nor trailing processes and changed their overall shape frequently. Comparison of the behavior of cells expressing GFP under the control of T alpha 1 tubulin or nestin promoter revealed that rapid motility was specific to cells in the neuronal lineage. The rapid movement was sensitive to an actin perturbing reagent and also dependent on the calcium influx through L-type calcium channels. These results indicate the presence of a specific form of precursor cell migration in the embryonic olfactory bulb.

Age Factors↗

PubMed2005

Determination of absolute protein numbers in single synapses by a GFP-based calibration technique.

To build a quantitative model of molecular organization of neurons, it is essential to have information about the number of protein molecules at individual synapses. Here we developed a method to estimate absolute numbers of individual proteins at actual excitatory synapses by calibrating the fluorescence intensity of microspheres with single EGFP molecules. In cultured hippocampal neurons, we observed a monotonous increase of postsynaptic protein numbers per single synapse during neuronal differentiation and subsequent stabilization. At maturity we calculated that a single excitatory postsynaptic site contains 100-450 of individual postsynaptic proteins, such as PSD-95, GKAP, Shank and Homer. This narrow range of postsynaptic protein content suggests relatively simple stoichiometry of postsynaptic molecular organization. The EGFP-based calibration technique provides an unprecedented general method for estimating the amounts of proteins in macromolecular complexes.

Animals↗

PubMed2005

High-purity lineage selection of embryonic stem cell-derived neurons.

The derivation of somatic cell types from pluripotent and self-renewing embryonic stem (ES) cells offers attractive prospects for basic research, compound development, and regenerative medicine. A key prerequisite for biomedical applications of ES cells is the ability to differentiate and isolate defined somatic cell populations at high purity. In this study, we explore the potential of the Talpha1- enhanced green fluorescent protein (EGFP) transgene and polysialic acid (PSA)-neural cell adhesion molecule (NCAM) as lineage selection markers for the derivation of ES cell-derived neurons. Upon controlled in vitro differentiation, ES cells engineered to express EGFP under control of the Talpha1-tubulin promoter exhibited exclusive transgene expression in neurons. Similarly, PSA-NCAM expression during the early stages of ES cell differentiation was restricted to neuronal progeny. Talpha1- EGFP- and PSA-NCAM-positive neurons comprised both inhibitory and excitatory phenotypes. Compared to Talpha1-EGFP, the expression of PSA-NCAM was initiated at slightly earlier stages of neural differentiation. FACSorting of Talpha1-EGFP-positive cells and immunopanning of PSA-NCAMexpressing cells yielded neuronal populations at purities up to 99.6% and 96.9%, respectively. These findings depict Talpha1-EGFP and PSA-NCAM as suitable markers for high-purity selection of early ES cell-derived neurons.

Animals↗

PubMed2005

Bi-directional regulation of postsynaptic cortactin distribution by BDNF and NMDA receptor activity.

Abstract Cortactin is an F-actin-associated protein which interacts with the postsynaptic scaffolding protein Shank at the SH3 domain and is localized within the dendritic spine in the mouse neuron. Green fluorescent protein (GFP)-based time-lapse imaging revealed cortactin redistribution from dendritic cytoplasm to postsynaptic sites by application of brain-derived neurotrophic factor (BDNF). This response was mediated by mitogen-activated protein (MAP) kinase activation and was dependent on the C-terminal SH3 domain. In contrast, activation of N-methyl-D-aspartate (NMDA) receptors induced loss of cortactin from postsynaptic sites. This NMDA-dependent redistribution was blocked by an Src family kinase inhibitor. Conversely, increasing Src family kinase activity induced cortactin phosphorylation and loss of cortactin from the postsynaptic sites. Finally, blocking of endogenous BDNF reduced the amount of cortactin at the postsynaptic sites and an NMDA receptor antagonist prevented this reduction. These results indicate the importance of counterbalance between BDNF and NMDA receptor-mediated signalling in the reorganization of the postsynaptic actin cytoskeleton during neuronal development.

2-Amino-5-phosphonovalerate↗

PubMed2004

Electroporation-mediated gene transfer system applied to cultured CNS neurons.

Electroporation is effective in transferring foreign genes into immature neurons in intact brain tissue. We utilized this approach to transfect genes into developing rodent hippocampi. Transfected hippocampi were subsequently dissociated and allowed to differentiate in culture. By optimizing developmental stage of the hippocampus, promoters to drive the marker cDNA, and culture conditions, neurons kept strong expression of multiple marker genes for more than two weeks after electroporation. We could also induce transient expression in mature neurons by combining electroporation of plasmids containing loxP-flanked stopper sequences and infection of Cre-producing recombinant adenoviruses. The system described here is useful in analyzing biological roles of multiple genes in specific stages of neuronal development.

Animals↗

PubMed2003

p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor signaling.

N-methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRepsilon2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRepsilon2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.

Actins↗

PubMed2003

Synchronized formation and remodeling of postsynaptic densities: long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with green fluorescent protein.

To explore mechanisms governing the formation and remodeling of postsynaptic density (PSD), we used dissociated cultures of hippocampal neurons isolated from transgenic embryos expressing green fluorescent protein (GFP)-tagged PSD proteins PSD-Zip45 (Homer 1c) and PSD-95. Expression of GFP-tagged PSD molecules was stable, and the remodeling process of PSDs could be followed for >1 week. A higher expression level of GFP-PSD-Zip45 enabled us to quantitatively analyze the amount of PSD-Zip45 clusters during development. Repetitive imaging of the same cell populations between 11 and 17 d in culture revealed an increase of the average PSD-Zip45 cluster density from 0.32 to 0.73/microm. Newly generated dendrites rapidly acquired GFP-PSD-Zip45 clusters, and their density reached the level of parental dendrites within a few days. Temporal profiles of GFP-PSD-Zip45 cluster density showed a variety of patterns. Some dendrites showed a monotonous increase of clusters, whereas others showed complex patterns, including short decremental stages. Analysis of long-term remodeling of PSD-95-GFP clusters confirmed that the decremental stages were not specific to the PSD-Zip45 clusters. Comparison of the temporal profiles of the cluster density among neurons indicated synchronization of both GFP-PSD-Zip45 and PSD-95 clustering within individual cells. Furthermore, activation of cAMP-dependent protein kinase suppressed the decremental stages of cluster remodeling. These observations suggest the presence of signaling mechanisms that can induce synchronized addition or elimination of PSD proteins throughout dendritic arborization of a single neuron.

Animals↗

PubMed2003

Synaptic targeting of PSD-Zip45 (Homer 1c) and its involvement in the synaptic accumulation of F-actin.

PSD-Zip45/Homer1c, which contains an enabled/VASP homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic density (PSD) scaffold protein that interacts with metabotropic glutamate receptors and the shank family. We studied the molecular mechanism underlying the synaptic targeting of PSD-Zip45 in cultured hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the extreme C-terminal leucine zipper motif markedly suppressed the synaptic localization of endogenous shank but not PSD-95 or GKAP. In contrast, an overexpressed GKAP mutant lacking shank binding activity had no effect on the synaptic localization of shank. Actin depolymerization by latrunculin A reduced the synaptic localization of PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression of PSD-Zip45 enhanced the accumulation of synaptic F-actin. Additionally, overexpression of PSD-Zip45 and an isoform of shank induced synaptic enlargement in association with the further accumulation of synaptic F-actin. The EVH1 domain and extreme C-terminal leucine zipper motif of PSD-Zip45 were also critical for these events. Thus, these data suggest that the PSD-Zip45-shank and PSD-95-GKAP complexes form different synaptic compartments, and PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic accumulation of F-actin.

Actins↗

PubMed2003

The dynamic organization of postsynaptic proteins: translocating molecules regulate synaptic function.

Physiological roles for postsynaptic molecules in synaptogenesis and plasticity are under intense investigation. Recent imaging experiments, including GFP-based and single-particle tracking strategies, reveal rapid movement of synaptic components to and from the postsynaptic sites. Furthermore, specific patterns of neuronal activity and/or activation of specific transmitter receptors trigger selective translocation of postsynaptic components. These emerging dynamic properties of synaptic specializations add another layer of complexity to the signaling mechanisms of CNS synapses.

Animals↗

PubMed2003

Migration and differentiation of neural progenitor cells from two different regions of embryonic central nervous system after transplantation into the intact spinal cord.

Transplantation of in vitro-expanded neural stem cells (NSCs) is a potentially powerful tool to repair functions of the injured spinal cord. A prerequisite for the successful transplantation therapy is identification of optimized experimental parameters that can promote maximal survival, extensive migration and selective differentiation of the transplanted NSC population in the spinal cord. To this end, we evaluated the basic characteristics of NSC-like cells from two different donor sources, the embryonic hippocampus and spinal cord, after transplantation into the neonatal spinal cord. Proliferation and differentiation phenotypes of both NSC-like cells can be controlled by the concentration of fibroblast growth factor-2 (FGF-2) in vitro. Both NSC-like cells can survive within the environment of the intact neonatal spinal cord and showed extensive migratory behaviour shortly after transplantation. However, quantitative analysis revealed preferential migration of hippocampus-derived cells in the dorsal white matter. Both NSC-like cells showed restricted phenotype toward the oligodendroglial lineage after transplantation. Transplantation of the mixture of two cell types revealed selective survival of hippocampus-derived NSC-like cells. This study indicates the possibility of transplanting hippocampus-derived NSCs to supply the cell source for immature oligodendrocytes, which are thought to be essential for both the myelination and trophic support of regenerating axons in the dorsal white matter of the spinal cord.

Animals↗

PubMed2002

Coexpression of a Ca(v)1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity.

L-type Ca(2) channels play a critical role in many types of cells, including nerve, muscle and endocrine cells. The most popular and effective tools for analyzing the roles of L-type calcium channels (L-channels) are specific antagonists such as dihydropyrigines. With these drugs however, it is difficult to target specific cells. One solution is to develop a genetically targetable inhibitor coded by DNA. As a candidate for such an inhibitor, a dominant negative mutant of Ca(v)1.2 was designed by mimicking an ascidian 3-domain-type alpha1 subunit (that inhibits the full-length subunit's current). The 3-domain-type Ca(v)1.2 subunit significantly inhibited wild-type Ca(v)1.2 current, but not other ionic currents such as Ca(v)2.1 and Na(v) channels in Xenopus oocyte expression systems. Western blot analysis showed that the expression of the wild-type protein into the plasma membrane was significantly suppressed on coexpression with the truncated protein. These findings support that an N-terminus-truncated mutant could serve as a specific genetically encoded inhibitor for L-channels.

Amino Acid Sequence↗