Yoshimi Hasegawa

Role of the Rho GTPase-activating protein RICS in neurite outgrowth.

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase‐activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS–/– neurons was higher than that in wild‐type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS–/– mice bore longer neurites than those from wild‐type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.

Calmodulin Overexpression Causes Ca2+-Dependent Apoptosis of Pancreatic β Cells, Which Can Be Prevented by Inhibition of Nitric Oxide Synthase

We investigated the mechanism of β-cell loss in transgenic mice with elevated levels of β cell calmodulin. The transgenic mice experienced a sudden rise in blood glucose levels between 21 and 28 days of age. This change was associated with development of severe hypoinsulinemia and loss of β cells from the islets. Ultrastructural analysis revealed that compromised granule formation and apoptotic changes in the transgenic β cells preceded the onset of hyperglycemia. Intraperitoneal injection of tolbutamide, an antidiabetic sulfonylurea, decreased blood glucose levels but increased the number of apoptotic β cells. Finally, injection of transgenic mice with Nω-nitro-l-arginine methyl ester, which inhibits nitric oxide synthase activity, prevented hyperglycemia and lessened the changes in number and size of β cells. Because immunofluorescent staining revealed preferential distribution of neural nitric oxide synthase in pancreatic β cells, we speculate that overexpression of calmodulin sensitizes the β cells to Ca2+-dependent activation of neural nitric oxide synthase, which mediates apoptosis.

Dopamine-degrading activity of monoamine oxidase is not detected by histochemistry in neurons of the substantia nigra pars compacta of the rat

Monoamine oxidase (MAO) activity was examined in neurons of the substantia nigra pars compacta (SNC) of the rat using a histochemical method, and compared to MAO activity in neurons of the locus coeruleus (LC) and dorsal raphe nucleus (DR). Using dopamine as a substrate, dopamine-degrading MAO activity was not detected in any SNC neurons, although LC and DR neurons were intensely stained for this activity. We further examined MAO activity in these neurons using other substrates, including serotonin (an MAO type A preferential substrate), beta-phenylethylamine (an MAO type B preferential substrate), and tyramine (a substrate common to both MAO types A and B). As for dopamine, no SNC neurons were stained for MAO activity using any of these other substrates. In contrast, LC neurons were intensely stained when either serotonin or tyramine was used, and DR neurons were darkly stained when either beta-phenylethylamine or tyramine was used. The lack of evidence of MAO activity in the SNC is surprising given that there are densely packed tyrosine hydroxylase (TH)-immunoreactive neurons in the SNC (i.e., dopaminergic neurons). By comparison, in the LC and DR the distribution patterns of the MAO-stained neurons were similar to those of TH-immunolabeled neurons (i.e., noradrenergic neurons) and serotonin-immunoreactive neurons, respectively. Our results suggest that dopamine-degrading MAO activity and MAO types A and B activities in SNC dopamine neurons are very low compared to MAO activity in LC noradrenaline neurons and in DR serotonin neurons.

Dopamine-degrading activity of monoamine oxidase in locus coeruleus and dorsal raphe nucleus neurons. A histochemical study in the rat

Dopamine-degrading activity of monoamine oxidase (MAO) was detected in the rat using a new histochemical method, with dopamine as the substrate. Our new method, designed to minimise the non-enzymatic oxidation of dopamine, was applied in combination with tyrosine hydroxylase (TH) and serotonin immunohistochemistry. We showed that the distribution pattern of MAO neurons was similar to that of TH-immunoreactive neurons (i.e. noradrenergic neurons) in the locus coeruleus (LC) and to that of serotonergic neurons in the dorsal raphe nucleus (DR). Since LC neurons form dopamine during noradrenaline biosynthesis, and DR neurons produce dopamine from exogenously administered L-dopa, our results indicate that dopamine produced in LC and DR neurons may be degraded, at least in part, by MAO.

Histochemical study of dopamine-degrading monoamine oxidase activity in dopaminergic neurons of rat brain

We examined whether dopamine-degrading activity of monoamine oxidase (MAO) is present in dopaminergic neurons of the rat brain. We employed a double-labeling procedure combining immunohistochemistry for tyrosine hydroxylase (TH) and enzyme histochemistry for MAO activity using dopamine as a substrate. The following dopaminergic cell groups were examined: A16 (glomerular layer of the olfactory bulb), A14 (hypothalamic periventricular region), A13 (zona incerta), A12 (arcuate nucleus), A11 (periventricular gray matter of the caudal thalamus), A10 (ventral tegmental area), A9 (substantia nigra pars compacta, SNC) and A8 (retrorubral nucleus). Although no MAO activity was detected in any of the TH-immunoreactive dopaminergic neurons, strong dopamine-degrading MAO activity was found in TH-positive neurons in the locus coeruleus (LC) (i.e., noradrenergic neurons). Our results indicate that dopamine-degrading MAO activity is very low in dopaminergic neurons compared to the MAO activity in LC noradrenergic neurons.

Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: Possible role in convergent extension

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1-/- mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1-/- mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1-/- mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes.

High expression of Pitx-2 in the ICAT-deficient metanephros leads to developmental arrest.

ICAT (Inhibitor of β-catenin and T cell factor) inhibits the interaction between β-catenin and TCF/LEF transcription factor and serves as a negative regulator of Wnt signaling. In a subset of ICAT knockout mice, significant delay in the ureteric bud branching and renal agenesis are observed. In order to examine the process of this developmental defect, molecular changes were analyzed in fetal ICAT–/– kidneys with a focus on Wnt-signaling associated factors. The protein level of active β-catenin was elevated in ICAT–/– kidneys. DNA microarray and immunohistochemical analyses revealed that the expression of a Wnt target gene Pitx-2 was enhanced in ICAT–/– kidneys. There was no genotypic difference in the expression level of another Wnt target gene, c-Ret. These results suggest that the enhancement of Pitx-2 expression induced by activated Wnt signaling leads to delays in ureteric bud branching and subsequent renal agenesis. In the ICAT–/– kidneys which developed to E18.5 without any apparent defect, renal glomeruli, convoluted tubules and collecting ducts were decreased in density and showed abnormal structure. ICAT may be required for various developmental stages during renal development.

Noradrenaline-degrading activity of monoamine oxidase is localized in noradrenergic neurons of the locus coeruleus of the rat

We found intense monoamine oxidase (MAO) activity in rat locus coeruleus (LC) neurons by means of a histochemical method using noradrenaline as a substrate. This MAO activity was abolished by clorgyline, a specific inhibitor of MAO type A. Fluorescence immunohistochemistry for tyrosine hydroxylase (TH) combined with MAO histochemistry revealed intense MAO activity in virtually all TH-immunoreactive LC neurons (i.e. noradrenergic neurons). The results indicate that noradrenaline produced in LC neurons might be degraded by MAO type A activity.

Dopamine-degrading activity of monoamine oxidase in serotonin and noradrenaline neurons

097 IMMUNOHISTOCHEMICAL ANALYSIS ON THE ROLE OF THE ADENOSINE Al RECEPTOR IN EPILEPSY TOMOYO OCHIISHI’, MASATOSHI TAKITA’, SHINYA S SUZUKI’, MITSUSHI IKEMOTO’, HIROYASLJ NAKATA 3 ‘Biosignalling Dept. and ‘Biomolecular Engineering Dept., Natnl. Inst. of Biosci. & Human-Technol., Tsukuba, Ibaraki 305 8566, 3 Mol. & Cell. Neurobiol., Tokyo Metropolitan Inst. for Neurosci., Fuchu Tokyo 183-8526, Japan

Phenotypic changes of AADC-only-immunoreactive cells in the alimentary canal of the laboratory shrew, Suncus murinus , induced by systemic administration of monoamine precursors

In order to elucidate the function of anti-aromatic acid decarboxylase (AADC)-only-positive cells in the alimentary canal, 5-hydroxy-L-tryptophan (5-HTP) or L-3,4-dihydroxyphenylalanine (L-DOPA) was intraperitoneally injected into the laboratory shrew, Suncus murinus, and immunohistochemical studies were conducted on continuous sections of the alimentary canal using specific antisera against tyrosine hydroxylase (TH), AADC, dopamine (DA), and serotonin (5-HT). AADC-only-positive cells localized to the epithelial layer of the alimentary canal from the stomach to the large intestine. These AADC-only-positive cells became DA- and AADC-positive cells after L-DOPA injection, and 5-HT- and AADC-positive cells after 5-HTP injection. These results strongly indicate that the AADC-only-positive cells in the alimentary canal of Suncus murinus are capable of synthesizing DA and 5-HT simultaneously upon administration of L-DOPA and 5-HTP.