Naoyuki Inagaki

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.

CRMP-2 induces axons in cultured hippocampal neurons.

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2–4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

Is the histaminergic neuron system a regulatory center for whole-brain activity?

Recent immunocytochemical studies have demonstrated the existence of histaminergic neurons in the brain, which are concentrated in the tuberomammillary nucleus of the posterior hypothalamus, and which project efferent fibers to almost all parts of the brain. Three subtypes of histamine receptors are widely distributed in the brain, not only on neurons but also on astrocytes and blood vessels. Consistent with its wide-ranging output, the histaminergic neuron system regulates various activities of the brain, such as the arousal state, brain energy metabolism, locomotor activity, neuroendocrine, autonomic and vestibular functions, feeding, drinking, sexual behavior, and analgesia--this regulation is possibly achieved by the histaminergic system as a whole.

Phosphorylation of collapsin response mediator protein-2 by Rho-kinase. Evidence for two separate signaling pathways for growth cone collapse.

We previously identified Rho-associated protein kinase (Rho-kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse was not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.

Phospholipase C-γ and Phosphoinositide 3-Kinase Mediate Cytoplasmic Signaling in Nerve Growth Cone Guidance

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.

BDNF and NT-3 induce intracellular Ca2+ elevation in hippocampal neurones

The effects of neurotrophins on intracellular Ca2+ levels in rat hippocampal neurones were studied in vitro using fura-2 fluorescence microscopy. BDNF and NT-3, but not NGF, rapidly increased cytoplasmic Ca2+ concentrations in these neurones ten-fold to reach 1 microM. Moreover in some of the neurones both BDNF and NT-3 elicited Ca2+ responses, indicative of the presence of functional receptors for these neurotrophins in the same cell. In these cultures approximately 80% of the hippocampal neurones were stained with antibodies against full-length TrkB. The expression of functional TrkB was also confirmed by RNA analysis. These results demonstrate the presence of functional receptors for BDNF and NT-3 in hippocampal neurones.

Circadian rhythm of histamine release from the hypothalamus of freely moving rats

Using an in vivo microdialysis technique coupled with HPLC-fluorometry, the release of neuronal histamine from the anterior hypothalamic area was monitored continuously in conscious, freely moving rats under a 12:12 h light:dark cycle. Spontaneous locomotor activity of the rats was measured simultaneously using a locomotor activity counter. Histamine release gradually increased in the second half of the light period (1400-2000) and the average histamine release during the dark period (2000-0800, 0.20 +/- 0.02 pmol/30 min) was significantly higher than that during the light period (0.12 +/- 0.01 pmol/30 min). This clear circadian change in the release suggests that the central histaminergic system is related to the circadian rhythm of rats.

Characterization of TrkB Receptor‐Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5). Here, we report that both truncated and full‐length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT‐4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT‐4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C‐γ (PLC‐γ) and SH2‐containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC‐γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival‐promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c‐ras in a concentration‐dependent manner. These results suggest that two different pathways, the c‐ras and the PLC‐γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.

Glial Fibrillary Acidic Protein: Dynamic Property and Regulation by Phosphorylation

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein of astroglia, and belongs to the type III subclass of IF proteins. IF proteins are composed of an amino‐terminal HEAD domain, a central ROD domain and a carboxyterminal TAIL domain. GFAP, with a molecular mass of ˜50 KDa, has the smallest HEAD domain among type III IF proteins. Despite its insolubility, GFAP is in dynamic equilibrium between assembled filaments and unassembled subunits, as demonstrated using fluo‐rescently labeled GFAP molecules. Like other IF proteins, assembly of GFAP is regulated by phosphory‐lation‐dephosphorylation of the HEAD domain by altering its charge. This regulation of GFAP assembly contributes to extensive remodeling of glial frameworks in mitosis. Another type III IF protein, vimentin, colocalizes with GFAP in immature, reactive or radial glia, thereby indicating that vimentin has an important role in the build up of the glial architecture.

In vivo release of neuronal histamine in the hypothalamus of rats measured by microdialysis

SummaryUsing an in vivo intracerebral microdialysis method coupled with an HPLC-fluorometric method, we investigated the extracellular level of endogenous histamine in the anterior hypothalamic area of urethaneanaesthetized rats. The basal rate of release of endogenous histamine in the anterior hypothalamic area measured by this method was 0.09 + 0.01 pmol/20 min. When the anterior hypothalamic area was depolarized by infusion of 100 mM K+ through the dialysis membrane or electrical stimulation at 200 μ A was applied through an electrode implanted into the ipsilateral tuberomammillary nucleus, histamine release increased to 175% and 188%, respectively, of the basal level. These increases were completely suppressed by removal of extracellular Ca2+. The basal release of histamine was also suppressed after infusion of 10−6 M tetrodotoxin or i.p. administration of 100 mg/kg of α-fluoromethylhistidine. On the other hand, 3-fold increase in the basal release was observed after i. p. administration of 5 mg/kg thioperamide. These results clearly indicate that both the basal and evoked release of histamine measured by our method are of neuronal origin.