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H Nawa

Publications and source records attributed to H Nawa.

At least 19 recordsLinked to original sources

Gene expression profiling of primate neocortex: molecular neuroanatomy of cortical areas.

One hundred years have passed since Brodmann's mapping of the mammalian neocortex. Solely on the basis of morphological observations, he envisaged the conservation and differentiation of cortical areal structures across various species. We now know that neurochemical, connectional and functional heterogeneity of the neocortex accompanies the morphological divergence observed in such cytoarchitectonic studies. Nevertheless, we are yet far from fully understanding the biological significance of this cortical heterogeneity. In this article, we review our past works on the gene expression profiling of the postnatal primate cortical areas, by quantitative real-time polymerase chain reaction (PCR), DNA array, differential display PCR and in situ hybridization analyses. These studies revealed both the overall homogeneity of gene expression across different cortical areas and the presence of a small number of genes that show markedly area-specific expression patterns. In situ hybridization showed that, among such genes, occ1 and retinol-binding protein (RBP) mRNAs are selectively expressed in the neuronal populations that seem to be involved in distinct neural processing such as sensory reception (for occ1) and associative function (for RBP). Such a molecular neuroanatomical approach has the promise to provide an important link between structure and function of the cerebral cortex.

Animals↗

ErbB1 receptor ligands attenuate the expression of synaptic scaffolding proteins, GRIP1 and SAP97, in developing neocortex.

Scaffolding proteins containing postsynaptic density-95/discs large/zone occludens-1 (PDZ) domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. Little is known, however, about what regulates the expression of PDZ proteins and how the levels of these proteins influence synaptic development. Here, we show that ligands for epidermal growth factor receptors (ErbB1) decrease a particular set of PDZ proteins and negatively influence synaptic formation or maturation. In short-term neocortical cultures, concentrations of epidermal growth factor and amphiregulin (2-9 pM) decreased the expression of glutamate receptor interacting protein 1 (GRIP1) and synapse-associated protein 97 kDa (SAP97) without affecting postsynaptic density-95 (PSD-95) levels and glial proliferation. In long-term cultures, epidermal growth factor treatment resulted in a decrease in the frequency of pan-PDZ-immunoreactive aggregates on dendritic processes. A similar activity on the same PDZ proteins was observed in the developing neocortex following epidermal growth factor administration to rat neonates. Immunoblotting revealed that administered epidermal growth factor from the periphery activated brain ErbB1 receptors and decreased GRIP1 and SAP97 protein levels in the neocortex. Laser-confocal imaging indicated that epidermal growth factor administration suppressed the formation of pan-PDZ-immunoreactive puncta and dispersed those structures in vivo as well. These findings revealed a novel negative activity of ErbB1 receptor ligands that attenuates the expression of the PDZ proteins and inhibits postsynaptic maturation in developing neocortex.

Adaptor Proteins, Signal Transducing↗

Transforming growth factor-alpha changes firing properties of developing neocortical GABAergic neurons by down-regulation of voltage-gated potassium currents.

Transforming growth factor-alpha (TGFalpha), a member of the epidermal growth factor family, has neurotrophic actions on postmitotic neurons. We examined the chronic effects of TGFalpha on the electrophysiological properties of one type of GABAergic neuron, identified by its bipolar morphology, in neocortical primary culture. Approximately 85% of the bipolar neurons were GABA-immunoreactive. In response to depolarizing current injection, the bipolar neurons usually showed tonic firing of action potential under control conditions. After treatment with TGFalpha (20 ng/ml) for 2 days, these neurons failed to generate trains of action potentials. Furthermore, the treatment altered the action potential waveforms of the bipolar neurons, including the duration and amplitude of the fast after-hyperpolarization, which implies a reduction in voltage-gated potassium currents. In contrast, TGFalpha did not affect the firing properties of pyramidal-shaped non-GABAergic neurons. Voltage-clamp recordings from the bipolar neurons indicated that chronic treatment with TGFalpha markedly decreased the current densities of slow delayed rectifier (IK) and transient voltage-gated potassium currents, whereas the treatment had no effect on voltage-gated sodium current and fast delayed rectifier potassium current densities. Reverse transcription-polymerase chain reaction analysis of potassium channel mRNA in the bipolar neurons revealed that the reduction in the IK current density was caused by Kv2.2 mRNA down-regulation. Thus, chronic treatment with TGFalpha down-regulated slow delayed rectifier and transient voltage-gated potassium currents, and in parallel, suppressed repetitive generation of action potentials in the cortical GABAergic neurons.

4-Aminopyridine↗

Brain-derived neurotrophic factor upregulates and maintains AMPA receptor currents in neocortical GABAergic neurons.

The regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex.

Animals↗

Neonatal perturbation of neurotrophic signaling results in abnormal sensorimotor gating and social interaction in adults: implication for epidermal growth factor in cognitive development.

Epidermal growth factor (EGF) and its structurally related proteins are implicated in the developmental regulation of various brain neurons, including midbrain dopaminergic neurons. There are EGF and EGF receptor abnormalities in both brain tissues and blood from schizophrenic patients. We administered EGF to neonatal rats to transiently perturb endogenous EGF receptor signaling and evaluated the neurobehavioral consequences. EGF-treatment-induced transient impairment in tyrosine hydroxylase expression. The animals grew normally, exhibited normal weight increase, glial growth, and gross brain structures, and later lost the tyrosine hydroxylase abnormality. During and after development, however, the rats began to display various behavioral abnormalities. Abnormal sensorimotor gating was apparent, as measured by deficits in prepulse inhibition of acoustic startle. Motor activity and social interaction scores of the EGF-treated animals were also impaired in adult rats, though not in earlier developmental stages. In parallel, there was a significant abnormality in dopamine metabolism in the brain stem of the adult animals. Gross learning ability appeared to be normal as measured by active avoidance. These behavioral alterations, which are often present in schizophrenic models, were ameliorated by subchronic treatment with clozapine. Although the molecular and/or physiologic background(s) of these behavioral abnormalities await further investigation, the results of the present experiment indicate that abnormal EGF receptor stimulation given during limited neonatal stages can result in severe and persistent cognitive/behavioral dysfunctions, which appear only in adulthood.

Animals↗

Phosphatidylinositol 3-kinase: a molecule mediating BDNF-dependent spatial memory formation.

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic plasticity such as long-term potentiation (LTP), a form of synaptic correlate of learning and memory. BDNF is also implicated in learning and memory. We have demonstrated that radial arm maze training in rats for spatial learning and memory results in a significant increase in the BDNF mRNA expression in the hippocampus. Moreover, antisense BDNF oligonucleotide treatment impaired not only acquisition, but also maintenance and/or recall of spatial memory in the maze. Although these results suggest a role of BDNF for spatial memory processes, the signal transduction mechanisms that mediate the actions of BDNF remain unknown. Here we show that phosphorylation of BDNF receptor tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI3-K) and Akt, a target of PI3-K, in the hippocampus increased in parallel with spatial memory formation. Moreover, an activation of translational processes was suggested in the hippocampus after the maze training. When spatial learning was inhibited by antisense BDNF oligodeoxynucleotide, the activation was diminished. Chronic treatment with PI3-K inhibitor wortmannin impaired spatial learning. Our findings suggested that activation of TrkB/PI3-K and protein synthesis signaling pathway by BDNF in the hippocampus is important for spatial memory.

Androstadienes↗

Brain-derived neurotrophic factor modulates glucagon secretion from pancreatic alpha cells: its contribution to glucose metabolism.

AIM: Brain-derived neurotrophic factor (BDNF) reduces plasma glucose levels in obese db/db diabetic mice and is speculated to produce its effects via the hypothalamus, the regulatory centre of satiety and the autonomic nervous system. The potential effect of BDNF directly on peripheral endocrine organs, however, remains to be clarified. Here we report the effects of BDNF on hormonal secretion from pancreatic islets of Langerhans using their isolated culture. METHODS AND RESULTS: In an immunohistochemical study, mouse pancreatic alpha cells were stained specifically with the anti-TrkB (a specific receptor for BDNF) antibody. After 7 days culture of isolated islets of the normal mouse pancreas, 10 ng/ml BDNF decreased the secretion of glucagon per 6 h significantly less than that of the control (p = 0.016). In contrast, there were no significant changes in insulin secretion or glucagon and insulin contents in the islets cultured under the same conditions. In vivo administration of BDNF (10 mg/kg/day) to normal mice for 7 days significantly decreased their food consumption (p < 0.05). The fasting plasma glucose levels were decreased on day 7 compared with day 1 more significantly in BDNF-treated mice (p = 0.043) than in pair-fed control mice (p = 0.14). In newborn BDNF-knockout mice, fasting plasma glucose levels increased in the order of homozygote, heterozygote and wild type (p = 0.033). No apparent immunohistochemical abnormality was observed for pancreatic glucagon in the BDNF-knockout mice. CONCLUSION: These data suggest that BDNF affects glucose metabolism not only with its anorectic effect but also with modulated glucagon secretion from pancreatic alpha cells.

Animals↗

Neurotrophins induce BDNF expression through the glutamate receptor pathway in neocortical neurons.

Neurotrophins jointly exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. However, the functional interactions of neurotrophins or mechanisms through which neurotrophins regulate each other are still not clear. In the present study, brain-derived neurotrophic factor (BDNF) mRNA expression is induced by neurotrophin-4/5 (NT-4/5) and by BDNF itself in neocortical neurons. K252a, a specific tyrosine kinase (Trk) inhibitor, completely suppresses BDNF- and NT-4/5-enhanced BDNF mRNA expression. NT-4/5 significantly augments BDNF protein production, which is also reversed by K252a. When neurons are incubated with neurotrophin-3 (NT-3) or nerve growth factor (NGF), there are no significant changes in BDNF mRNA or protein expression. Interestingly, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or the N-methyl-D-aspartate (NMDA) receptor blocker AP-5 completely suppresses NT-4/5-enhanced BDNF protein production, while tetrodotoxin (TTX) only suppresses NT-4/5-enhanced BDNF production by 50%. Additionally, the mitogen activated protein (MAP) kinase inhibitor PD98059 enhances BDNF-induced glutamate receptor-1 (GluR1) protein expression, but a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 strongly reduces BDNF-induced GluR1 protein expression. Taken together, glutamate receptors are important for the regulation of BDNF expression by neurotrophins, and MAP and PI3K kinases differentially modulate AMPA receptor expression in the cortical neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Abnormal expression of epidermal growth factor and its receptor in the forebrain and serum of schizophrenic patients.

Epidermal growth factor (EGF) comprises a structurally related family of proteins containing heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGFalpha) that regulates the development of dopaminergic neurons as well as monoamine metabolism. We assessed the contribution of EGF to schizophrenia by measuring EGF family protein levels in postmortem brains and in fresh serum of schizophrenic patients and control subjects. EGF protein levels were decreased in the prefrontal cortex and striatum of schizophrenic patients, whereas the levels of HB-EGF and TGFalpha were not significantly different in any of the regions examined. Conversely, EGF receptor expression was elevated in the prefrontal cortex. Serum EGF levels were markedly reduced in schizophrenic patients, even in young, drug-free patients. Chronic treatment of animals with the antipsychotic drug haloperidol had no influence on EGF levels in the brain or serum. These findings suggest that there is abnormal EGF production in various central and peripheral tissues of patients with both acute and chronic schizophrenia. EGF might thus provide a molecular substrate for the pathologic manifestation of the illness, although additional studies are required to determine a potential link between impaired EGF signaling and the pathology/etiology of schizophrenia.

Adult↗

Triphasic vascular responses to bradykinin in the mesenteric resistance artery of the rat.

The vascular effects of bradykinin were studied in rat perfused mesenteric vascular beds with active tone. Bolus injections of bradykinin (1-1000 pmol) but not des-Arg(9)-bradykinin (bradykinin B(1) receptor agonist) induced triphasic vascular responses: the initial sharp vasodilation followed by transient vasoconstriction and subsequent gradual vasodilation. The triphasic vascular responses to bradykinin were abolished by FR 172357 (3-bromo-8-[2,6-dichloro-3-[N-[(E)-4-(N,N-dimethylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-metylimidazo[1,2-a]pyridine) (bradykinin B(2) receptor antagonist, 0.1 microM). Endothelium removal with sodium deoxycholate and N(w)-nitro-L-arginine (300 microM) abolished the bradykinin-induced initial sharp vasodilation. Indomethacin (0.5 microM) and seratrodast (thromboxane A(2) receptor antagonist, 0.5 and 5 microM) abolished the bradykinin-induced second vasoconstriction. The bradykinin-induced third vasodilation was abolished by capsaicin (1 microM) and calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist, 0.5 microM). These findings suggest that the bradykinin-induced initial sharp vasodilation is endothelium dependent, that endogenous thromboxane A(2) is involved in the second vasoconstriction, and that the third slow vasodilation is produced by activation of capsaicin-sensitive CGRP-containing nerves.

Animals↗

Brain-derived neurotrophic factor enhances neuronal translation by activating multiple initiation processes: comparison with the effects of insulin.

The effects of neurotrophic factors on translational activation were investigated in cortical neurons. Brain-derived neurotrophic factor (BDNF) increased protein synthesis within 30 min, whereas insulin produced a weaker enhancement of protein synthesis. BDNF-triggered protein synthesis was inhibited by LY294002, PD98059, and rapamycin, whereas the effect of insulin was unaffected by PD98059. To explore the mechanisms underlying this effect, the protein phosphorylation cascades that lead to the activation of translation initiation in neurons were examined. BDNF induced the phosphorylation of both eukaryote initiation factor (eIF) 4E and its binding protein (eIF4E-binding protein-1). The former reaction was inhibited by PD98059, whereas the latter was inhibited by LY294002 or rapamycin. In agreement, BDNF induced the phosphorylation of mammalian TOR (target of rapamycin) and enhanced its kinase activity toward eIF4E-binding protein-1. In contrast, insulin failed to activate MAPK and did not induce the phosphorylation of eIF4E. Since BDNF and insulin increased the activity of eIF2B and eIF2, the only difference between them was eIF4E phosphorylation. Thus, this may explain the lower activity of insulin in potentiating neuronal protein synthesis. These results suggest strongly that BDNF simultaneously activates multiple signaling cascades consisting of phosphatidylinositol 3-kinase, mammalian TOR, and MAPK to enhance translation initiation in neurons.

Animals↗

N-methyl-D-aspartate-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor down-regulation involves interaction of the carboxyl terminus of GluR2/3 with Pick1. Ligand-binding studies using Sindbis vectors carrying AMPA receptor decoys.

The dynamics of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-d-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 microm NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [(3)H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [(3)H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1.

Animals↗

A quantitative study on the expression of synapsin II and N-ethylmaleimide-sensitive fusion protein in schizophrenic patients.

The application of DNA array technology to schizophrenic studies enabled us to assess molecular features of this disease. The expression of synapsin II and N-ethylmaleimide-sensitive fusion protein (NSF) mRNAs is reported to decrease in the prefrontal cortex of these patients. We attempted to reproduce this result with two distinct approaches. With high quality samples, mRNA and protein levels for synapsin II and NSF were measured by real-time polymerase chain reaction and by immunoblotting. Both experiments led to the same conclusion: The expression of these presynaptic markers is not altered significantly in the prefrontal cortex of our schizophrenic samples, compared to that in control subjects. These observations suggest that the neurochemical impairments of synapses reported in schizophrenia are not evident for all presynaptic markers and needs to be re-evaluated at molecular levels.

Adult↗

Adrenergic nerves mediate acetylcholine-induced endothelium-independent vasodilation in the rat mesenteric resistance artery.

Mechanisms underlying acetylcholine-induced endothelium-independent vasodilation were studied in the rat mesenteric vascular bed isolated from Wistar rats. In preparations without endothelium, and contracted by perfusion with Krebs solution containing methoxamine (2-7 microM), perfusion of acetylcholine (1-100 microM) for 1 min produced a concentration-dependent vasodilation. Denervation of denuded preparations by cold storage (4 degrees C for 72 h) abolished the acetylcholine-induced vasodilation; 10 and 100 nM atropine abolished 1 and 10 microM acetylcholine-induced vasodilation, but it inhibited only 20% of vasodilation by 100 microM acetylcholine. The acetylcholine-induced atropine-resistant vasodilation was inhibited by 10 and 100 microM hexamethonium, 5 microM guanethidine, 50 microM bretylium, in vitro 6-hydroxydopamine (2 mM for 20 min, twice), 1 microM capsaicin and 0.5 microM calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist). These findings suggest that the acetylcholine-induced endothelium-independent nicotinic vasodilation requires the presence of intact adrenergic nerves, and is mediated by endogenous CGRP released from CGRP-containing nerves.

Acetylcholine↗

Biochemical evidence for localization of AMPA-type glutamate receptor subunits in the dendritic raft.

A low density Triton-insoluble fraction with characteristic lipid composition was prepared from synaptic plasma membrane from the rat forebrain. The fraction was named dendritic raft based on its absence of the presynaptic marker synaptophysin, the presence of postsynaptic Glutamate receptor (GluR) subunits, and its resemblance to raft, caveolae-like structure. We found a differential distribution of NMDA-type and AMPA-type GluR subunits in the dendritic raft and postsynaptic density (PSD) fractions; the latter type GluR subunits were localized to the dendritic raft as well as PSD fraction, whereas the former type was mostly localized to the PSD fraction. We also found the differential distribution of the components of ras/mitogen-activated protein kinase (MAPK) pathway to the dendritic raft and PSD fractions. Dendritic raft and PSD may possibly interact at the postsynaptic sites for efficient signal processing that is required for expression of synaptic plasticity.

Animals↗

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons.

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.

Animals↗

Similarity and variation in gene expression among human cerebral cortical subregions revealed by DNA macroarrays: technical consideration of RNA expression profiling from postmortem samples.

The functional regionality of the human cerebral cortex suggests that a set of genes might be activated in each subregion of the neocortex to support its specific functions. To test this hypothesis, we employed the DNA array technique to compare the mRNA expression profiles of three neocortical subregions of the human brain: prefrontal cortex (Area 46), motor cortex (Area 4) and visual cortex (Area 17). The macroarray analysis on high quality mRNA from postmortem brains revealed that the expression profiles of the different cortical areas are almost similar: only six out of 1088 known genes exhibited significant differences (>2-fold) in their expression. RT-PCR studies with an increased number of samples confirmed that expression of only two genes, annexin II and early growth response protein 1, varied by 2-fold among the regions, whereas expression of the others showed large inter-individual difference. These results suggest that the whole neocortex of humans is more homogeneous than we expected at the level of gross gene expression profiles. In parallel, sensitivity and accuracy of radioisotope-based DNA macroarrays and fluorescence-based DNA microarrays were tested.

Aged↗

Characterization of a novel synGAP isoform, synGAP-beta.

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.

Aging↗