Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Investigation of radical scavenging effects of acetaminophen, p-aminophenol and their O-sulfated conjugates.

Acetaminophen (APAP) and p-aminophenol (p-AP) are the analogous simple phenolic compounds that undergo sulfate conjugation (sulfation) by cytosolic sulfotransferases. Sulfation is generally thought to lead to the inactivation and disposal of endogenous as well as xenobiotic compounds. This study aimed to investigate the antioxidative effects of O-sulfated form of APAP and p-AP, i.e., APAPS and p-APS, in comparison with their unsulfated counterparts. Using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, the antioxidant capacity of APAPS was shown to be approximately 126-times lower than that of APAP. In contrast, p-APS displayed comparable activity as unsulfated p-AP. Similar trends concerning the suppressive effects of these chemicals on cellular O2- radical generation were found using an activated granulocytic neutrophil cell model. Collectively, these results indicated that, depending on the presence of an additional "active site", sulfation may not always decrease the antioxidant activities of phenolic compounds.

PGAM5 interacts with Bcl-rambo and regulates apoptosis and mitophagy.

Bcl-rambo, also known as BCL2L13, has been reported to regulate apoptosis, mitochondrial fragmentation, and mitophagy. However, the molecular mechanisms by which Bcl-rambo regulates these processes currently remain unclear. In the present study, we identified phosphoglycerate mutase member 5 (PGAM5) as an emerging partner interacting with Bcl-rambo through phenotypic Drosophila screening. The rough eye phenotype induced by human Bcl-rambo was partly rescued by the knockdown of pgam5-2, a mammalian ortholog of PGAM5. Bcl-rambo bound to PGAM5, and their interaction required the Bcl-rambo transmembrane domain. The co-expression of Bcl-rambo and PGAM5 promoted effector caspase activity in human embryonic kidney 293T cells. The transient overexpression of Bcl-rambo increased LC3B-II levels, which had been decreased by the co-expression of PGAM5. These results suggest that PGAM5 promotes Bcl-rambo-dependent apoptosis, but conversely interferes with Bcl-rambo-dependent mitophagy.

Brivaracetam prevents astroglial l-glutamate release associated with hemichannel through modulation of synaptic vesicle protein.

The antiepileptic/anticonvulsive action of brivaracetam is considered to occur via modulation of synaptic vesicle protein 2A (SV2A); however, the pharmacological mechanisms of action have not been fully characterised. To explore the antiepileptic/anticonvulsive mechanism of brivaracetam associated with SV2A modulation, this study determined concentration-dependent effects of brivaracetam on astroglial L-glutamate release associated with connexin43 (Cx43), tumour-necrosis factor-α (TNFα) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor of rat primary cultured astrocytes using ultra-high-performance liquid chromatography. Furthermore, interaction among TNFα, elevated extracellular K+ and brivaracetam on expression of SV2A and Cx43 was determined using capillary immunoblotting. TNFα and elevated extracellular K+ predominantly enhanced astroglial L-glutamate release associated with respective AMPA/glutamate receptor and hemichannel. These effects were enhanced by a synergistic effect of TNFα and elevated extracellular K+ in combination. The activation of astroglial L-glutamate release, and expression of SV2A and Cx43 in the plasma membrane was suppressed by subchronic brivaracetam administration but were unaffected by acute administration. These results suggest that migration of SV2A to the astroglial plasma membrane by hyperexcitability activates astroglial glutamatergic transmission, perhaps via hemichannel activation. Subchronic brivaracetam administration suppressed TNFα-induced activation of AMPA/glutamate receptor and hemichannel via inhibition of ectopic SV2A. These findings suggest that combined inhibition of vesicular and ectopic SV2A functions contribute to the antiepileptic/anticonvulsive mechanism of brivaracetam action.

Effects of Carbamazepine, Lacosamide and Zonisamide on Gliotransmitter Release Associated with Activated Astroglial Hemichannels.

Recent studies using the genetic partial epilepsy model have demonstrated that hyperfunction of astroglial hemichannels contributes to pathomechanism of epileptic seizure. Therefore, to explore the novel anticonvulsive mechanisms, the present study determined the effects of voltage-dependent Na+ channel (VDSC)-inhibiting anticonvulsants, carbamazepine (CBZ), lacosamide (LCM), and zonisamide (ZNS) on the astroglial release of l-glutamate and adenosine triphosphate (ATP). The effects of subchronic administration of therapeutic-relevant dose of three anticonvulsants on the release of l-glutamate and ATP in the orbitofrontal cortex (OFC) were determined using microdialysis. The concentration-dependent effects of acute and subchronic administrations of anticonvulsants on astroglial gliotransmitter release were determined using primary cultured astrocytes. The concentration-dependent effects of subchronic administrations of anticonvulsants on connexin43 (Cx43) expression in the plasma membrane of primary cultured astrocytes were determined using the Simple Western system. An increase in the levels of extracellular K+ resulted in a concentration-dependent increase in the astroglial release of l-glutamate and ATP. The depleted levels of extracellular Ca2+ alone did not affect astroglial gliotransmitter release but did accelerate K+-evoked gliotransmitter release via activation of astroglial hemichannels. Both non-selective hemichannel inhibitor carbenoxolone (CBX) and selective Cx43 inhibitor GAP19 prevented both gliotransmitter release through activated astroglial hemichannels and the hemichannel-activating process induced by elevation of the levels of extracellular K+ with depletion of the levels of extracellular Ca2+. ZNS subchronically decreased Cx43 expression and acutely/subchronically inhibited Cx43 hemichannel activity. LCM acutely inhibited hemichannel activity but did not subchronically affect Cx43 expression. Therapeutic-relevant concentration of CBZ did not affect hemichannel activity or Cx43 expression, but supratherapeutic concentration of CBZ decreased Cx43 expression and hemichannel activity. Therefore, the present study demonstrated the distinct effects of CBZ, LCM, and ZNS on gliotransmitter release via modulation of astroglial hemichannel function. The different features of the effects of three VDSC-inhibiting anticonvulsants on astroglial transmission associated with hemichannels, at least partially, possibly contributing to the formation of the properties of these three anticonvulsants, including the antiepileptic spectrum and adverse effects regarding mood and cognitive disturbance.

Pharmacological Discrimination of Effects of MK801 on Thalamocortical, Mesothalamic, and Mesocortical Transmissions.

N-methyl-d-aspartate/glutamate receptor (NMDAR) is one of the major voltage-sensitive ligand-gated cation channel. Several noncompetitive NMDAR antagonists contribute to pathophysiology of schizophrenia and mood disorders; however, the effects of inhibition of NMDAR on several transmitter system have not been well clarified. Thus, this study determined the selective NMDAR antagonist, MK801 (dizocilpine), on thalamocortical, mesothalamic, and mesocortical transmissions associated with l-glutamate, GABA, serotonin, norepinephrine, and dopamine using multiprobe microdialysis. Perfusion with MK801 into the medial prefrontal cortex (mPFC) increased and decreased respective regional releases of monoamine and GABA without affecting l-glutamate. The mPFC MK801-induced monoamine release is generated by the regional GABAergic disinhibition. Perfusion with MK801 into the reticular thalamic nucleus (RTN) decreased GABA release in the mediodorsal thalamic nucleus (MDTN) but increased releases of l-glutamate and catecholamine without affecting serotonin in the mPFC. The RTN MK801-induced l-glutamate release in the mPFC was generated by GABAergic disinhibition in the MDTN, but RTN MK801-induced catecholamine release in the mPFC was generated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/glutamate receptor (AMPAR) which received l-glutamate release from thalamocortical glutamatergic terminals in the mPFC. Perfusion with MK801 into the dorsal raphe nucleus (DRN) decreased GABA release in the DRN but selectively increased serotonin release in the MDTN and mPFC. These DRN MK801-induced serotonin releases in the both mPFC and MDTN were also generated by GABAergic disinhibition in the DRN. These results indicate that the GABAergic disinhibition induced by NMDAR inhibition plays important roles in the MK801-induced releases of l-glutamate and monoamine in thalamic nuclei and cortex.

Lurasidone Sub-Chronically Activates Serotonergic Transmission via Desensitization of 5-HT1A and 5-HT7 Receptors in Dorsal Raphe Nucleus.

Lurasidone is an atypical mood-stabilizing antipsychotic agent with unique receptor-binding profile, including 5-HT7 receptor (5-HT7R) antagonism. Effects of 5-HT7R antagonism on transmitter systems of schizophrenia and mood disorders, however, have not been well clarified. Thus, this study examined the mechanisms underlying the clinical effects of lurasidone by measuring mesocortical serotonergic transmission. Following systemic and local administrations of lurasidone, MK801 and 5-HT receptor modulators, we determined releases of 5-HT in dorsal raphe nucleus (DRN), mediodorsal thalamic nucleus (MDTN) and medial prefrontal cortex (mPFC) and γ-aminobutyric acid (GABA) in DRN using multiprobe microdialysis with ultra-high-performance liquid chromatography (UHPLC). Serotonergic and GABAergic neurons in the DRN are predominantly regulated by inhibitory 5-HT1A receptor (5-HT1AR) and excitatory 5-HT7R, respectively. Lurasidone acutely generates GABAergic disinhibition by 5-HT7R antagonism, but concomitant its 5-HT1AR agonism prevents serotonergic hyperactivation induced by 5-HT7R inhibition. During treatments with 5-HT1AR antagonist in DRN, lurasidone dose-dependently increased 5-HT release in the DRN, MDTN and mPFC. Contrary, lurasidone chronically enhanced serotonergic transmission and GABAergic disinhibition in the DRN by desensitizing both 5-HT1AR and 5-HT7R. These effects of lurasidone acutely prevented MK801-evoked 5-HT release by GABAergic disinhibition via N-methyl-D-aspartate (NMDA)/glutamate receptor (NMDA-R)-mediated inhibition of 5-HT1AR function, but enhanced MK801-induced 5-HT release by desensitizing 5-HT1AR and 5-HT7R. These results indicate that acutely lurasidone fails to affect 5-HT release, but chronically enhances serotonergic transmission by desensitizing both 5-HT1AR and 5-HT7R. These unique properties of lurasidone ameliorate the dysfunctions of NMDA-R and augment antidepressive effects.

Carbamazepine Attenuates Astroglial L-Glutamate Release Induced by Pro-Inflammatory Cytokines via Chronically Activation of Adenosine A2A Receptor.

Carbamazepine (CBZ) binds adenosine receptors, but detailed effects of CBZ on astroglial transmission associated with adenosine receptor still need to be clarified. To clarify adenosinergic action of CBZ on astroglial transmission, primary cultured astrocytes were acutely or chronically treated with CBZ, proinflammatory cytokines (interferon γ (IFNγ) and tumor necrosis factor α (TNFα)), and adenosine A2A receptor (A2AR) agonist (CGS21680). IFNγ and TNFα increased basal, adenophostin-A (AdA)-evoked, and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)-evoked astroglial L-glutamate releases. In physiological condition, CGS21680 increased basal astroglial L-glutamate release but glutamate transporter inhibition prevented this CGS21680 action. CBZ did not affect basal release, whereas glutamate transporter inhibition generated CBZ-induced glutamate release. Furthermore, AdA-evoked and AMPA-evoked releases were inhibited by CBZ but were unaffected by CGS21680. Contrary to physiological condition, chronic administrations of IFNγ and TNFα enhanced basal, AdA-, and AMPA-evoked releases, whereas IFNγ and TNFα decreased and increased CGS21680-evoked releases via modulation A2AR expression. Both chronic administration of CGS21680 and CBZ suppressed astroglial L-glutamate release responses induced by chronic cytokine exposer. Especifically, chronic administration of CBZ and CGS21680 prevented the reduction and elevation of A2AR expression by respective IFNγ and TNFα. These findings suggest that A2AR agonistic effects of CBZ contribute to chronic prevention of pathomechanisms developments of several neuropsychiatric disorders associated with proinflammatory cytokines.

Increased phenolic content and antioxidant capacity of the heated leaves of yacon (Smallanthus sonchifolius).

We investigated the content of phenolic compounds and antioxidant capacity of two batches of non-heated and heated leaves of the yacon cultivar "Andes no yuki", grown in Japan. Lyophilized yacon leaves heated at 160°C for 20 min and 100°C for 60 min had a 1.96 to 9.69-times higher total phenolic content than that of the non-heated leaves. Heated leaves exhibited a 1.98 to 4.07-times higher antioxidant capacity than that of the non-heated leaves in three different free radical scavenging assays. Heated leaves were more efficient at attenuating the superoxide anion radical production in human granulocytic cells than the non-heated leaves. High-performance liquid chromatography analysis revealed that, in the heated leaves, the caffeic acid content was 2.13 to 3.64-times higher and the chlorogenic acid content was slightly lower than those in the non-heated leaves. Hence, heat processing may affect the active constituent contents in yacon leaves, potentiating its antioxidant capacity.Abbreviations: ABTS+: 2,2'-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; DPPH: 1,1-diphenyl-2-picrylhydrazyl, HPLC: high-performance liquid chromatography; NBT: nitroblue tetrazolium; O2-: superoxide anion; PMA: phorbol 12-myristate 13-acetate; PMS: phenazine methosulfate; TEAC: Trolox equivalent antioxidant capacity.

Lurasidone inhibits NMDA receptor antagonist-induced functional abnormality of thalamocortical glutamatergic transmission via 5-HT7 receptor blockade.

BACKGROUND AND PURPOSE: Lurasidone is an atypical mood-stabilizing antipsychotic with a unique receptor-binding profile, including 5-HT7 receptor antagonism; however, the detailed effects of 5-HT7 receptor antagonism on various transmitter systems relevant to schizophrenia, particularly the thalamo-insular glutamatergic system and the underlying mechanisms, are yet to be clarified. EXPERIMENTAL APPROACH: We examined the mechanisms underlying the clinical effects of lurasidone by measuring the release of l-glutamate, GABA, dopamine, and noradrenaline in the reticular thalamic nucleus (RTN), mediodorsal thalamic nucleus (MDTN) and insula of freely moving rats in response to systemic injection or local infusion of lurasidone or MK-801 using multiprobe microdialysis with ultra-HPLC. KEY RESULTS: Systemic MK-801 (0.5 mg·kg-1 ) administration increased insular release of l-glutamate, dopamine, and noradrenaline but decreased GABA release. Systemic lurasidone (1 mg·kg-1 ) administration also increased insular release of l-glutamate, dopamine, and noradrenaline but without affecting GABA. Local lurasidone administration into the insula (3 µM) did not affect MK-801-induced insular release of l-glutamate or catecholamine, whereas local lurasidone administration into the MDTN (1 µM) inhibited MK-801-induced insular release of l-glutamate and catecholamine, similar to the 5-HT7 receptor antagonist SB269970. CONCLUSIONS AND IMPLICATIONS: The present results indicate that MK-801-induced insular l-glutamate release is generated by activation of thalamo-insular glutamatergic transmission via MDTN GABAergic disinhibition resulting from NMDA receptor inhibition in the MDTN and RTN. Lurasidone inhibited this MK-801-evoked insular l-glutamate release through inhibition of excitatory 5-HT7 receptor in the MDTN. These effects on thalamo-insular glutamatergic transmission may contribute to the antipsychotic and mood-stabilizing actions of lurasidone.

Memantine protects thalamocortical hyper-glutamatergic transmission induced by NMDA receptor antagonism via activation of system xc.

Deficiencies in N-methyl-d-aspartate (NMDA)/glutamate receptor (NMDAR) signaling have been considered central to the cognitive impairments of schizophrenia; however, an NMDAR antagonist memantine (MEM) improves cognitive impairments of Alzheimer's disease and schizophrenia. These mechanisms of paradoxical clinical effects of NMDAR antagonists remain unclear. To explore the mechanisms by which MK801 and MEM affect thalamocortical transmission, we determined interactions between local administrations of MK801, MEM, system xc- (Sxc), and metabotropic glutamate receptors (mGluRs) on extracellular glutamate and GABA levels in the mediodorsal thalamic nucleus (MDTN) and medial prefrontal cortex (mPFC) using dual-probe microdialysis with ultra-high-pressure liquid chromatography. Effects of MK801 and MEM on Sxc activity were also determined using primary cultured astrocytes. Sxc activity was enhanced by MEM, but was unaffected by MK801. MK801 enhanced thalamocortical glutamatergic transmission by GABAergic disinhibition in the MDTN. In the MDTN and the mPFC, MEM weakly increased glutamate release by activating Sxc, whereas MEM inhibited thalamocortical glutamatergic transmission. Paradoxical effects of MEM were induced following secondary activation of inhibitory II-mGluR and III-mGluR by exporting glutamate from astroglial Sxc. The present results suggest that the effects of therapeutically relevant concentrations of MEM on thalamocortical glutamatergic transmission are predominantly caused by activation of Sxc rather than inhibition of NMDAR. These demonstrations suggest that the combination between reduced NMDAR and activated Sxc contribute to the neuroprotective effects of MEM. Furthermore, activation of Sxc may compensate for the cognitive impairments that are induced by hyperactivation of thalamocortical glutamatergic transmission following activation of Sxc/II-mGluR in the MDTN and Sxc/II-mGluR/III-mGluR in the mPFC.

Effects of acute and sub-chronic administrations of guanfacine on catecholaminergic transmissions in the orbitofrontal cortex.

The selective α2A adrenoceptor agonist guanfacine reduces hyperactivity and improves cognitive impairment in patients with attention-deficit/hyperactivity disorder (ADHD). The major mechanisms of guanfacine have been considered to involve activation of postsynaptic α2A adrenoceptor in frontal pyramidal neurons. However, the effects of chronic guanfacine administration on catecholaminergic transmissions associated with the orbitofrontal cortex (OFC) remain unclear. To explore the mechanisms of action of guanfacine on catecholaminergic transmission, the effects of its acute local or sub-chronic systemic administration on catecholamine release within pathways from locus coeruleus (LC) to OFC and reticular thalamic nucleus (RTN), from RTN to mediodorsal thalamic nucleus (MDTN), and from MDTN to OFC were determined using multi-probe microdialysis with ultra-high performance liquid chromatography. Acute OFC local administration of guanfacine did not affect catecholamine release in OFC. Acute LC local and sub-chronic systemic administrations of guanfacine reduced norepinephrine release in LC, OFC and RTN, and also reduced GABA release in MDTN, whereas AMPA-induced (perfusion with AMPA into NDTN) releases of l-glutamate, norepinephrine and dopamine in OFC were enhanced by sub-chronic systemic guanfacine administration. This study identified that catecholaminergic transmission is composed of three pathways: direct noradrenergic and co-releasing catecholaminergic LC-OFC pathways and intermediate LC-OFC (LC-RTN-MDTN-OFC) pathway. We demonstrated the dual actions of guanfacine on catecholaminergic transmission: attenuation of direct noradrenergic LC-OFC transmission at the resting stage and enhancement of direct co-releasing catecholaminergic LC-OFC transmission via GABAergic disinhibition in the intermediate LC-OFC pathway. These dual actions of guanfacine probably contribute to clinical actions of guanfacine against ADHD and its comorbid symptoms. This article is part of the Special Issue entitled 'Current status of the neurobiology of aggression and impulsivity'.

Involvement of sonic hedgehog and notch signaling in regenerative neurogenesis in adult zebrafish optic tectum after stab injury.

Unlike humans and other mammals, adult zebrafish have the superior capability to recover from central nervous system (CNS) injury. We previously found that proliferation of radial glia (RG) is induced in response to stab injury in optic tectum and that new neurons are generated from RG after stab injury. However, molecular mechanisms which regulate proliferation and differentiation of RG are not well known. In the present study, we investigated Shh and Notch signaling as potential mechanisms regulating regeneration in the optic tectum of adult zebrafish. We used Shh reporter fish and confirmed that canonical Shh signaling is activated specifically in RG after stab injury. Moreover, we have shown that Shh signaling promotes RG proliferation and suppresses their differentiation into neurons after stab injury. In contrast, Notch signaling was down-regulated after stab injury, indicated by the decrease in the expression level of her4 and her6, a target gene of Notch signaling. We also found that inhibition of Notch signaling after stab injury induced more proliferative RG, but that inhibition of Notch signaling inhibited generation of newborn neurons from RG after stab injury. These results suggest that high level of Notch signaling keeps RG quiescent and that appropriate level of Notch signaling is required for generation of newborn neurons from RG. Under physiological condition, activation of Shh signaling or inhibition of Notch signaling also induced RG proliferation. In adult optic tectum of zebrafish, canonical Shh signaling and Notch signaling play important roles in proliferation and differentiation of RG in physiological and regenerative conditions.

Unique antioxidant effects of herbal leaf tea and stem tea from Moringa oleifera L. especially on superoxide anion radical generation systems.

This study aimed to investigate the unique antioxidative effects of Japanese moringa products, herbal leaf tea and stem tea, using established free radical assays, focusing on superoxide anion (O2-) radical generation systems. Hot-water extracts from moringa teas resulted in different but lower scavenging activities than Trolox in four synthetic free radical models. Interestingly, these extracts further showed higher O2- radical scavenging effects than Trolox in the phenazine methosulfate-NADH-nitroblue tetrazolium and xanthine oxidase assay systems. Incubating human neutrophils in the presence of these tea extracts rather than Trolox effectively suppressed cellular O2- radical generation. Among the eight known phenolic constituents of moringa leaves, caffeic acid and chlorogenic acid may be responsible for the O2-specific radical scavenging capacity stronger than that of Trolox. These results suggest that moringa herbal teas are a good source of natural antioxidants for preventing O2- radical-mediated disorders. Abbreviations: O2-: superoxide anion; ROS: reactive oxygen species; H2O2: hydrogen peroxide; XOD: xanthine oxidase; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ABTS+: 2,2'-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; CPZ+: chlorpromazine cation; PMS: phenazine methosulfate; NBT: nitroblue tetrazolium; PMA: phorbol 12-myristate 13-acetate.

Radical scavenging effects of 1-naphthol, 2-naphthol, and their sulfate-conjugates.

1-Naphthol (1-Nap) and 2-naphthol (2-Nap) are phenolic isomers that may be subjected to sulfate conjugation in vivo. Phase-II sulfate conjugation of phenolic compounds is generally thought to result in their inactivation. This study aimed to investigate the antioxidative effects of 1-NapS and 2-NapS, in comparison with their unsulfated counterparts, using established free radical scavenging assays. Based on the calculated EC50 values, 1-NapS resulted in 5.60 to 7.35-times lower antioxidative activity than 1-Nap. In contrast, 2-NapS showed comparable activities as did the unsulfated 2-Nap. Collectively, the results obtained indicated that sulfate conjugation of the Nap isomers did not always result in the decrease of their antioxidant activity, and the antioxidant activity that remained appeared to depend on the position of sulfation.

Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish.

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial-like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up-regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3ß inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.

Wnt and Shh signals regulate neural stem cell proliferation and differentiation in the optic tectum of adult zebrafish.

Adult neurogenesis occurs more commonly in teleosts, represented by zebrafish, than in mammals. Zebrafish is therefore considered a suitable model to study adult neurogenesis, for which the regulatory molecular mechanisms remain little known. Our previous study revealed that neuroepithelial-like neural stem cells (NSCs) are located at the edge of the dorsomedial region. We also showed that Notch signaling inhibits NSC proliferation in this region. In the present study, we reported the expression of Wnt and Shh signaling components in this region of the optic tectum. Moreover, inhibitors of Wnt and Shh signaling suppressed NSC proliferation, suggesting that these pathways promote NSC proliferation. Shh is particularly required for maintaining Sox2-positive NSCs. Our experimental data also indicate the involvement of these signaling pathways in neural differentiation from NSCs. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1206-1220, 2017.

Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays, Especially on Different Superoxide Anion Radical Generation Systems.

UNLABELLED: Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot-water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot-water extract was similarly demonstrated among these extracts. Varying concentrations of the hot-water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC50 28.1 µg/mL), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (EC50 23.7 µg/mL), galvinoxyl radical (EC50 3.06 µg/mL), and chlorpromazine cation radical (EC50 475 µg/mL). The yacon tea-leaf extract further demonstrated superoxide anion (O2(-)) radical scavenging effects in the phenazine methosulfate-NADH-nitroblue tetrazolium (EC50 64.5 µg/mL) and xanthine oxidase assay systems (EC50 20.7 µg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea-leaf extract could suppress the cellular O2(-) radical generation (IC50 65.7 µg/mL) in a phorbol 12-myristate 13-acetate-activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders. PRACTICAL APPLICATION: Yacon has been considered to be a potent alternative food source for patients who require a dietary cure in regional area, while the leaf part has been provided and consumed as an herbal tea in local markets. We demonstrated here potent antioxidative effects of the tea leaves from yacon in different free radical assays, reducing power assay, and cellular superoxide anion radical generation assay. Results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders.

Intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic brain injury estimated using the microdialysis-electron spin resonance method.

AIM: The intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic (HI) brain injury was estimated using the microdialysis-electron spin resonance method. MATERIAL AND METHODS: Seven-day-old Wistar rats were subjected to a modified Levine's procedure for producing HI brain injury. After HI insult, pups were returned and reared with their dams. Seven weeks after HI insult, their intracerebral antioxidant abilities were measured using the microdialysis-electron spin resonance method after the intraperitoneal injection of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Ascorbic acid, L-cysteine, and glutathione (GSH) were also determined. The rats without HI insult were used as a control. RESULTS: The decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in the non-ligated side of the cerebral hemisphere of the HI group was significantly larger than that of the control group. The amounts of ascorbic acid in the perfusate from the non-ligated side of the HI group were about four times larger than those of the control group. The amounts of L-cysteine and GSH of the HI group were about 10 times larger than those of the control group. CONCLUSIONS: The antioxidant ability in the non-ligated sides of the cerebral hemispheres of the mature rats 7 weeks after neonatal HI insult was higher than that of the control group. Higher amounts of ascorbic acid and GSH supported the higher antioxidant ability. The increase of the intracerebral antioxidant ability of the non-ligated side indicates the compensation of motor function for the lost side. The present results should offer important insights into the prognosis for hypoxic-ischemic encephalopathy.

Involvement of the orexin system in adrenal sympathetic regulation.

Orexin (hypocretin) is a neuropeptide secreted from hypothalamic neurons that is known to be activated during motivated behaviors and active waking. Presently, our knowledge of orexin is mainly limited to the central nervous system, and the involvement of the orexin system in peripheral tissues has received little attention. In the present study, we analyzed the existence of the orexin system in the adrenal medulla, which is part of the sympathetic nervous system. Orexin and its receptors are expressed in the bovine adrenal medulla. Orexins stimulated intracellular calcium changes and epinephrine release from cultured bovine adrenal medullary cells. Applied orexin decreased expression of prepro-orexin, orexin receptor-1 and orexin receptor-2, suggesting negative feedback regulation in the adrenal gland. Our results indicate involvement of the orexin system in the sympathetic regulation of the adrenal medulla.