Sadaaki Maeda

Phenotypic Characterization of Transgenic Mice Overexpressing Neuregulin-1

Background Neuregulin-1 (NRG1) is one of the susceptibility genes for schizophrenia and implicated in the neurotrophic regulation of GABAergic and dopaminergic neurons, myelination, and NMDA receptor function. Postmortem studies often indicate a pathologic association of increased NRG1 expression or signaling with this illness. However, the psychobehavioral implication of NRG1 signaling has mainly been investigated using hypomorphic mutant mice for individual NRG1 splice variants. Methodology/Principal Findings To assess the behavioral impact of hyper NRG1 signaling, we generated and analyzed two independent mouse transgenic (Tg) lines carrying the transgene of green fluorescent protein (GFP)-tagged type-1 NRG1 cDNA. The promoter of elongation-factor 1α gene drove ubiquitous expression of GFP-tagged NRG1 in the whole brain. As compared to control littermates, both heterozygous NRG1-Tg lines showed increased locomotor activity, a nonsignificant trend toward decreasing prepulse inhibition, and decreased context-dependent fear learning but exhibited normal levels of tone-dependent learning. In addition, social interaction scores in both Tg lines were reduced in an isolation-induced resident-intruder test. There were also phenotypic increases in a GABAergic marker (parvalbumin) as well as in myelination markers (myelin basic protein and 2′,3′-cyclic nucleotide 3′-phosphodiesterase) in their frontal cortex, indicating the authenticity of NRG1 hyper-signaling, although there were marked decreases in tyrosine hydroxylase levels and dopamine content in the hippocampus. Conclusions These findings suggest that aberrant hyper-signals of NRG1 also disrupt various cognitive and behavioral processes. Thus, neuropathological implication of hyper NRG1 signaling in psychiatric diseases should be evaluated with further experimentation.

Effects of taste stimulation on β-endorphin levels in rat cerebrospinal fluid and plasma.

Opioids are suggested to be involved in generation of palatability and facilitation of consumption of food and fluid. We measured the level of an endogenous opioid, beta-endorphin, in the cerebrospinal fluid (CSF) and plasma after free drinking of water and taste solutions in Wistar rats. When the water-deprived animals were allowed to drink 10 mL of water, the level of beta-endorphin increased significantly 60 and 90 min after the start of drinking in both samples. beta-Endorphin in the CSF increased most after ingestion of 0.5 M sucrose and 0.005 M saccharin followed by 0.1 M NaCl, 0.1 mM quinine and water. An intragastric infusion of 7 mL of water did not change the beta-endorphin level. Essentially the same results were obtained for plasma samples except that NaCl and quinine solutions did not increase beta-endorphin levels. Sucrose became ineffective in releasing beta-endorphin in both samples after the establishment of conditioned taste aversions to this taste stimulus. These results suggest that the release of beta-endorphin is positively correlated with the palatability of taste stimuli, and that CSF beta-endorphin also reflects the reinforcement of fluid intake in thirsty animals.

Apelin Is a Crucial Factor for Hypoxia-Induced Retinal Angiogenesis

Objective—To investigate the role of endogenous apelin in pathological retinal angiogenesis. Methods and Results—The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein–coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway. Conclusion—These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca2+/calmodulin‐dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+‐containing medium after exposure to Ca2+‐free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+‐mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge‐induced injury in a dose‐dependent manner (10−10–10−8M). Cyclosporin A at 1 µM mimicked the effect of FK506. Rapamycin (1 µM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge‐induced increase in cytosolic Ca2+ level nor Na+‐Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Involvement of endoplasmic reticulum stress on the cell death induced by 6-hydroxydopamine in human neuroblastoma SH-SY5Y cells.

Endoplasmic reticulum (ER) dysfunction is known to activate the unfolded protein response, which is characterized by the activation of two divergent processes, i.e., suppression of the initiation process in global protein synthesis and expression of glucose-regulated protein 78 (Bip/Grp78) and the C/EBP homologous transcription factor CHOP/Gadd153. In this study, we examined the expression of CHOP/Gadd153 and Bip/Grp78 in human neuroblastoma SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), which is used to prepare animal models of Parkinson’s disease. 6-OHDA treatment induced cell death, in a concentration-dependent manner, which was inhibited by co-treatment with an antioxidant N-acetylcysteine. 6-OHDA was also effective in decreasing proteasome activity and in increasing the levels of high molecular ubiquitin-conjugated proteins. Furthermore, 6-OHDA induced a marked increase in the expression of both CHOP/Gadd153 and Bip/Grp78. This increase was prevented by N-acetylcysteine. Taken together, our data indicate that ER dysfunction is at least in part involved in the mechanisms underlying cell death induced by 6-OHDA in SH-SY5Y cells.

Nitric Oxide Protects Macrophages from Hydrogen Peroxide-Induced Apoptosis by Inducing the Formation of Catalase

We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2-treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, l-buthionine-(S,R)-sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production of catalase.

Nitric oxide at a low concentration protects murine macrophage RAW264 cells against nitric oxide‐induced death via cGMP signaling pathway

We investigated the cytoprotective effect of low‐dose nitric oxide (NO) on NO‐induced cell death in mouse macrophage‐like cell line RAW264. Sodium nitroprusside (SNP), an NO donor, at a high concentration (4 mM) released cytochrome c from mitochondria and induced death in RAW264 cells. Acetyl‐L‐aspartyl‐L‐glutamyl‐L‐valyl‐L‐aspart‐1‐al (Ac‐DEVD‐CHO, 100–200 μM), a caspase‐3 inhibitor, attenuated the SNP‐induced cell death in a concentration‐dependent manner. Pretreatment with 100 μM SNP for 24 h, which had no effect on cell viability, attenuated the cell death and reduced cytochrome c release from mitochondria to the cytosol induced by 4 mM SNP. LY83583 (1–3 μM) and 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ, 30–100 μM), soluble guanylate cyclase inhibitors, negated the protective effect of the 100 μM SNP pretreatment. Pretreatment with 1 mM dibutylyl guanosine‐3′,5′‐cyclic monophosphate (DBcGMP), a cell‐permeable guanosine‐3′,5′‐cyclic monophosphate (cGMP) analogue, for 24 h inhibited both cytochrome c release and cell death induced by SNP. Protein kinase G inhibitor KT5823 (10 μM) significantly reduced the cytoprotective effects of low‐dose SNP and DBcGMP. These results indicate that low‐dose NO protects RAW264 cells from NO‐induced apoptosis through cGMP production and activation of protein kinase G.

Insulin-like growth factor-1 protects peroxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation.

We investigated the effect of IGF‐1 on cell death induced by peroxynitrite in human neuroblastoma SH‐SY5Y cells. Exposure of the cells to 3‐morpholinosydnonimine (SIN‐1), a peroxynitrite donor, caused cytochrome c release from the mitochondria, caspase‐3‐like activation, and cell death. Pre‐incubation of the cells with the caspase‐3 inhibitor partially prevented SIN‐1‐induced cell death. Simultaneous addition of IGF‐1 reduced SIN‐1‐induced caspase‐3‐like activation and cell death, whereas IGF‐1 failed to reduce the release of cytochrome c. IGF‐1 increased Akt phosphorylation, and Akt phosphorylation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3‐kinase. In addition, wortmannin prevented IGF‐1‐evoked inhibition of cell death and caspase‐3‐like activation. In a cell‐free system, addition of cytochrome c to cytosolic fraction resulted in caspase‐3‐like activation. The activation was reduced when the cytosolic fraction prepared from IGF‐1‐treated cells was used. These results suggest that IGF‐1 protects peroxynitrite‐induced cell death downstream of cytochrome c release through the inhibition of caspase‐3‐like activation. J. Cell. Biochem. 84: 708–716, 2002.

Intracerebroventricular treatment of mice with pertussis toxin induces hyperalgesia and enhances 3H-nitrendipine binding to synaptic membranes: Similarity with morphine tolerance

SummaryThe effect of intracerebroventricular treatment of mice with pertussis toxin (PTX) on pain perception and 3H-nitrendipine binding was examined to study a possible change in the GTP-binding proteins in morphine tolerant rodents. It was observed that both PTX treatment and chronic administration of morphine cause hyperalgesia in the acetic acid-induced writhing test. Analgesic effects brought by the acute administration of morphine or nifedipine, a calcium antagonist, were not affected by PTX treatment. In synaptic membrane fractions prepared from mice treated with PTX or morphine chronically, specific binding of 3H-nitrendipine was enhanced approximately 41.8% and 35.7%, respectively, without alteration in its affinity. Chronic administration of morphine followed by PTX treatment did not display further increases in 3H-nitrendipine binding.These results suggest that the PTX-sensitive GTP-binding proteins may not be involved in the manifestation of the analgesic effect of morphine in mice.

Morphine prevents peroxynitrite-induced death of human neuroblastoma SH-SY5Y cells through a direct scavenging action

N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12), a nitric oxide donor, 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite (ONOO-), and peroxynitrite induced cell death accompanied by DNA fragmentation in human neuroblastoma SH-SY5Y cell cultures. Morphine prevented the cell death induced by SIN-1 or peroxynitrite, but not that induced by NOC12. The protective effect of morphine was concentration-dependent (10-100 microM), but was not antagonized by naloxone. The selective ligands for opioid receptor subtypes, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO, micro-opioid receptor agonist), [D-Pen2,5]enkephalin (DPDPE, delta-opioid receptor agonist) and trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benze neacetamide (U-50488, kappa-opioid receptor agonist) even at the concentration of 100 microM did not prevent the cell death induced by SIN-1. From measurement of the absorbance spectrum of peroxynitrite, the decomposition of peroxynitrite in 0.25 M potassium phosphate buffer (pH 7.4) was very rapid and complete within seconds. However, the absorbance was very stable in the presence of morphine. In addition, morphine inhibited peroxynitrite-induced nitration of tyrosine in a concentration-dependent manner. These results indicate that morphine rapidly reacts with peroxynitrite. The present study showed that morphine prevented peroxynitrite-induced cell death through its direct scavenging action, suggesting that morphine can protect cells against damage caused by peroxynitrite.