Miyuki Tauchi

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.

Distribution of glucagon-like peptide-1 immunoreactivity in the hypothalamic paraventricular and supraoptic nuclei

Glucagon-like peptide-1 (GLP-1) plays a role in modulating neuroendocrine and autonomic function. The hypothalamic paraventricular nucleus (PVN) contains aggregations of GLP-1 fibers and expresses GLP-1 receptors, making it a likely site of action for GLP-1 signaling. The current study was designed to establish domains of GLP-1 action, focusing on axosomatic appositions on different neuroendocrine and autonomic cell populations in the PVN. The data indicate abundant GLP-1-immunoreactive terminal appositions on corticotropin-releasing hormone neurons in the medial parvocellular PVN. GLP-1 positive boutons can also be observed in apposition to oxytocinergic neurons and on retrogradely labeled pre-autonomic neurons projecting to the region of the nucleus of the solitary tract. In contrast, there were very few vasopressinergic neurons with GLP-1 appositions. Overall, the data indicate that the central GLP-1 system preferentially targets neurons in hypophysiotrophic zones of the PVN, consistent with excitatory actions of GLP-1 on adrenocorticotropin release. GLP-1 is also in position to influence oxytocin secretion and control outflow to brainstem cardiovascular relays.

Glucocorticoid regulation of preproglucagon transcription and RNA stability during stress.

Stress elicits a synchronized response of the endocrine, sympathetic, and central nervous systems to preserve homeostasis and well-being. Glucagon-like peptide-1 (GLP-1), a primary posttranslational product of the preproglucagon (PPG) gene, activates both physical and psychological stress responses. The current study examined mechanisms regulating expression of PPG gene products in the hindbrain. Our results indicate that PPG mRNA decreases rapidly after exposure to acute stressors of multiple modalities. Reduced mRNA levels are accompanied by reduced GLP-1 immunoreactivity in the paraventricular nucleus of hypothalamus, suggesting release at PPG terminals. Stress-induced decrements in PPG mRNA were attenuated in adrenalectomized-corticosterone-replaced rats, suggesting that mRNA down-regulation is due at least in part to glucocorticoid secretion. In contrast, acute stress increased levels of PPG heteronuclear RNA (hnRNA) in a glucocorticoid-dependent manner, suggesting that decreases in PPG mRNA are due to increased degradation rather than reduced transcription. Glucocorticoid administration to unstressed rats is sufficient to cause decrements in PPG mRNA and increments in PPG hnRNA. These findings suggest that glucocorticoids deplete the pool of transcribed PPG mRNA and concurrently stimulate PPG gene transcription, with the latter allowing a mechanism for replenishment of PPG mRNA after stress cessation. The combination of rapid PPG mRNA depletion and initiation of PPG transcription within 30 min is consistent with a rapid action of glucocorticoids on GLP-1 bioavailability, resulting in a transient reduction in the capacity for neuropeptidergic excitation of stress responses.

ROLE OF CENTRAL GLUCAGON-LIKE PEPTIDE-1 IN HYPOTHALAMO-PITUITARY-ADRENOCORTICAL FACILITATION FOLLOWING CHRONIC STRESS

Central glucagon-like peptide-1 (GLP-1) regulates food intake, glucose homeostasis, and behavioral and neuroendocrine responses to acute stress. Given its pronounced role in acute stress regulation, the GLP-1 system is a prime candidate for mediating the prolonged drive of the hypothalamo-pituitary-adrenocortical axis by chronic stress. To test this hypothesis, we evaluated the necessity and sufficiency of GLP-1 for production of chronic stress-induced changes in HPA axis function. Exogenous GLP-1 or the GLP-1 receptor antagonist, dHG-exendin, were delivered into the 3rd ventricle of control animals or animals exposed to chronic variable stress (CVS) for 7 days. Animals in the CVS groups received GLP-1 or dHG-exendin immediately prior to each stress exposure. Prior to and at the end of the 7-day trial, chronically-stressed animals were subjected to a novel stressor to test for HPA axis facilitation. Neither GLP-1 nor dHG-exendin affected CVS-associated increases in adrenal weight or decreases in basal plasma glucose levels. In addition, neither exogenous GLP-1 nor dHG-exendin altered any index of HPA axis activity in unstressed rats. However, GLP-1 enhanced CVS-induced facilitation of corticosterone (but not ACTH) response to an acute stress, whereas dHG-exendin inhibited facilitation. In addition, GLP-1 decreased body weight in chronically-stressed animals. dHG-exendin increased food intake and body weight in unstressed animals, consistent with a tonic role for GLP-1 in body weight regulation. Overall, our data suggest that brain GLP-1 modulates HPA axis activity within the context of chronic stress, perhaps at the level of the adrenal gland.

Suppression of dendritic cell functions contributes to the anti-inflammatory action of granulocyte-colony stimulating factor in experimental stroke

Cerebral ischemia provokes an inflammatory cascade, which is assumed to secondarily worsen ischemic tissue damage. Linking adaptive and innate immunity dendritic cells (DCs) are key regulators of the immune system. The hematopoietic factor G-CSF is able to modulate DC-mediated immune processes. Although G-CSF is under investigation for the treatment of stroke, only limited information exists about its effects on stroke-induced inflammation. Therefore, we investigated the impact of G-CSF on cerebral DC migration and maturation as well as on the mediated immune response in an experimental stroke model in rats by means of transient middle cerebral artery occlusion (tMCAO). Immunohistochemistry and quantitative PCR were performed of the ischemic brain and flow cytometrical analysis of peripheral blood. G-CSF led to a reduction of the infarct size and an improved neurological outcome. Immunohistochemistry confirmed a reduced migration of DCs and mature antigen-presenting cells after G-CSF treatment. Compared to the untreated tMCAO group, G-CSF led to an inhibited DC activation and maturation. This was shown by a significantly decreased cerebral transcription of TLR2 and the DC maturation markers, CD83 and CD86, as well as by an inhibition of stroke-induced increase in immunocompetent DCs (OX62⁺OX6⁺) in peripheral blood. Cerebral expression of the proinflammatory cytokine TNF-α was reduced, indicating an attenuation of cerebral inflammation. Our data suggest an induction of DC migration and maturation under ischemic conditions and identify DCs as a potential target to modulate postischemic cerebral inflammation. Suppression of both enhanced DC migration and maturation might contribute to the neuroprotective action of G-CSF in experimental stroke.

Different treatment settings of Granulocyte-Colony Stimulating Factor and their impact on T cell-specific immune response in experimental stroke.

BACKGROUND Cerebral ischemia is associated with infectious complications due to immunosuppression and decreased T lymphocyte activity. G-CSF, which has neuroprotective properties, is known to modulate inflammatory processes after induced stroke. The aim of our study was to investigate the impact of G-CSF in experimental stroke and to compare two different modes of treatment, focusing on circulating T lymphocytes. METHODS Cerebral ischemia was induced in Wistar rats by occlusion of the middle cerebral artery, followed by reperfusion after 1h. G-CSF was applied either as a single dose 30 min after occlusion, or daily for seven days. Silver staining was used to determine infarct size. T lymphocytes in the peripheral blood were measured before and 7 days after induced cerebral ischemia by flow cytometry. In addition, migration of CD3-expressing T lymphocytes into the brain was investigated by immunohistochemistry. RESULTS Both single dose and daily treatment with G-CSF significantly reduced infarct size. A significant improvement of neurological outcome was only observed after single application of G-CSF. While a decrease in peripheral T lymphocytes was detected seven days after induced stroke, no reduction was observed in the G-CSF-treated groups. Apart from that, G-CSF significantly reduced the number of brain migrated T lymphocytes in both treatment settings as compared to vehicle. CONCLUSION A single dose of G-CSF exerted neuroprotective effects in ischemic stroke, which were less pronounced after daily G-CSF application. Both treatment strategies inhibited stroke-induced reduction of T lymphocytes in peripheral blood, which may have contributed to the reduction of infarct size.