Emi Iwata

Pergolide scavenges both hydroxyl and nitric oxide free radicals in vitro and inhibits lipid peroxidation in different regions of the rat brain

The free radical hypothesis for the pathogenesis and/or progression of Parkinsons disease (PD) has gained wide acceptance in recent years. Although it is clear that dopamine (DA) agonists cannot completely replace levodopa therapy, they can be beneficial early in the course of PD by reducing the accumulation of DA which undergoes auto-oxidation and generates cytotoxic free radicals. In the present study we demonstrate that pergolide, a widely used DA agonist, has free radical scavenging and antioxidant activities. Using a direct detection system for nitric oxide radical (NO.) by electron spin resonance (ESR) spectrometry in an in vitro .NO-generating system, we examined the quenching effects of pergolide on the amount of NO. generated. Pergolide dose-dependently scavenged NO.. In the competition assay, the IC50 value for pergolide was estimated to be about 30 microM. Pergolide also dose-dependently attenuated the hydroxyl radical (.OH) signal in an in vitro FeSO4-H2O2 ESR system with an approximate IC50 value of 300 microM. Furthermore, this agent significantly inhibited phospholipid peroxidation of rat brain homogenates in in vitro experiments and after repeated administration (0.5 mg/kg/24 h, i.p. for 7 days). Our findings suggest a neuroprotective role for pergolide on dopaminergic neurons due to its free radical scavenging and antioxidant properties.

Late-onset lipid peroxidation and neuronal cell death following transient forebrain ischemia in rat brain

We previously reported that iron deposition was seen in the cerebral cortex and hippocampal CA1 area late after transient forebrain ischemia generated by four-vessel occlusion in rats. Iron deposition in the hippocampal CA1 area was coupled with delayed pyramidal cell death, while that in the cerebral cortex was not accompanied by neuronal death or atrophy until 6 months after ischemia. Iron is involved in the formation of free radicals, thus contributing to lipid peroxidation. To elucidate whether this iron has deleterious effects on neurons, we investigated changes in the levels of lipid peroxidation and resulting neuronal damage in this ischemia model. The level of malondialdehyde plus 4-hydroxynonenal as major decomposition products of lipid peroxidation, monitored for 6 months beginning just after 30 min of transient forebrain ischemia, was significantly increased in the cerebral cortex at 6 months, and in the striatum from 1 week to 6 months compared to that in sham-operated controls. Histological changes were also examined up to 1 year after reperfusion by immunohistochemical methods. In contrast with the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy until 1 year after the ischemic insult. We showed that lipid peroxidation took place not only immediately after ischemia-reperfusion but also late after the ischemic insult in regions where iron was deposited, and we showed that neuronal cell death in the cerebral cortex appeared extremely late, suggesting that iron-mediated lipid peroxidation may be of importance in some slowly progressive forms of neurodegeneration.

Different effects of oxidative stress on activation of transcription factors in primary cultured rat neuronal and glial cells

We compared the cytotoxic effects of oxidative stress on neuronal and glial cells in vitro by examining the cell viability and changes in DNA-binding activities of transcription factors, AP-1 and CREB, using Trypan blue exclusion and electrophoretic mobility shift assay (EMSA), respectively. Neurotoxin 6-hydroxydopamine (6-OHDA) and H2O2 reduced the viability of both types of cells in time- and concentration-dependent manner. Both neurotoxins dose-dependently decreased DNA-binding activities in neuronal cells. The results of cell viability assay suggested that these changes may reflect the reduction in neuronal cell viability. In contrast, both reagents increased DNA-binding activities in glial cells, although they decreased cell numbers. These results suggest that the effects of oxidative stress on transcription factors is different in neuronal and glial cells. We also examined the effect of brain-derived neurotrophic factor (BDNF) on 6-OHDA- or H2O2-induced changes in DNA-binding activities. In neuronal cells, pre-treatment with BDNF prevented the decrease in DNA-binding activities induced by 6-OHDA or H2O2. In glial cells, the effect of BDNF on oxidative stress-induced changes in DNA-binding activities in the 6-OHDA-treated group were opposite to those in H2O2-treated group. Our results suggest that 6-OHDA and H2O2 may exert their cytotoxic mechanisms through different signal transduction systems.

Effects of single cyclosporin A pretreatment on pentylenetetrazol-induced convulsion and on TRE-binding activity in the rat brain

Using electrophoretic mobility-shift assay (EMSA), we examined changes in DNA-binding activities of transcriptional factor-activated protein-1 (AP-1), which is a Fos-Jun protein complex, onto its responsive element TRE in the hippocampus and amygdaloid nucleus of rats stimulated with pentylenetetrazol (PTZ) injection, and also investigated the effects of a single administration of the immunosuppressant cyclosporin A (CsA). In EMSA with nuclear extracts from the rat brain, the TRE-binding activity of AP-1 in the hippocampus and amygdaloid nucleus markedly increased 2 h after the PTZ injection (75 mg/kg, i.p.). These PTZ-induced increases of the TRE-binding protein in these regions were completely suppressed, by pretreatment with CsA (5 mg/kg, s.c.) 1 h before the PTZ injection. In addition, the administration of CsA significantly ameliorated PTZ-induced convulsion. This therapeutic effect of single CsA pretreatment may be based, in part, on the effects on the TRE-binding activity of AP-1 in the brain. Since single pretreatment of CsA in the present study had no effect on the PTZ-induced induction of c-fos mRNA, c-jun mRNA, Fos protein nor Jun protein, the inhibitory effects of single CsA administration on PTZ-induced TRE-binding activity in the brain may be related to the effects of CsA on AP-1 itself. These results suggest that an immune response via activation of transcriptional factor in the brain tissue is involved in the convulsion.

Cyclosporin A prevents ischemia-induced reduction of muscarinic acetylcholine receptors with suppression of microglial activation in gerbil hippocampus

We previously reported the late onset reduction of muscarinic acetylcholine receptors (LORMAR) which begins 7 days after a 5-min period of experimentally induced forebrain ischemia in the gerbil hippocampus. This study demonstrated that post-ischemic administration of cyclosporin A (CsA) reduced LORMAR 10 days after 5 min of forebrain ischemia in the gerbil hippocampus, suggesting that immunosuppression by CsA may reduce damage to the cholinergic system after ischemia. Microglia positive for HLA-DR class II antigen which presented in the hippocampal CA1 area, the region most vulnerable to ischemia, were also reduced by CsA. CsA may suppress microglial activation especially with regard to the antigen-presenting function, and LORMAR may be attenuated by this modulation of microglial function.

Protective effects of nicergoline against hydrogen peroxide toxicity in rat neuronal cell line

We examined the effects of nicergoline on hydrogen peroxide (H2O2)-induced neurotoxicity in cultured rat neuronal cell line (B50). H2O2 induced death of B50 cells in a dose-dependent manner. The H2O2-induced neuronal cell death was significantly decreased in B50 cells maintained in the presence of nicergoline. We compared the levels of antioxidants (glutathione, catalase and superoxide dismutase) in nicergoline-treated and untreated B50 cells. Lipid peroxidation products (thiobarbituric acid reactive substances, TBARS) levels were also measured. Cultures treated with nicergoline had higher levels of catalase activity. TBARS level was significantly lower in nicergoline-treated cells than in untreated cells. Our results suggest that nicergoline may induce the up-regulation of intracellular antioxidant defences and protect the neuronal cells against oxidative stress.

Age-related changes in composition of transcription factor, AP-1 complex in the rat brain

We examined age-related changes in composition of transcription factor, activator protein-1 (AP-1) which binds to TPA responsive element (TRE) in the non-stimulated rat brain, using electrophoretic mobility-shift assay with immunodepletion/supershift assay. The total TRE-binding activity in the frontal cortex and the hippocampus of the aged rats markedly decreased to 66% and 43%, respectively, and TRE-bindings of AP-1 in both regions also decreased to 82% and 66%, respectively, with aging. Jun-Jun dimers accounted for approximately half of the total TRE-bindings and 80-90% of the AP-1 bindings, while there were fewer Fos-Jun dimers, in both examined regions of the non-stimulated adult. The proportion of active Fos-Jun heterodimers in the frontal cortex increased to up to half of the AP-1 bindings in the aged rats, indicating that cortical AP-1-related transcription may increase with aging even under the non-stimulated condition. In the hippocampus, inactive Jun-Jun homodimers became predominant in AP-1 with aging. This regional diversity of age-related changes in the composition of AP-1 in the brain may be related to changes or dysfunction in neuronal signal transduction in the aged.

Involvement of protein kinase C in Ca2+-signaling pathways to activation of AP-1 DNA-binding activity evoked via NMDA- and voltage-gated Ca2+ channels

Abstract: Stimulation of cultured cerebellar granule cells with N‐methyl‐d‐aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein‐1 (AP‐1) DNA‐binding activity, which can be monitored by an increase in 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐responsive element (TRE)‐binding activity, in concert with c‐fos induction. For this increase in TRE‐binding activity, Ca2+ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca2+ chelator, BAPTA‐AM, abolished this increase. Close correspondence between the dose‐response curves of 45Ca2+ uptake and TRE‐binding activity by NMDA or KA suggested that Ca2+ influx not only triggered sequential activation of Ca2+‐signaling processes leading to the increase in TRE‐binding activity, but also controlled its increased level. Stimulation of non‐NMDA receptors by KA mainly caused Ca2+ influx through voltage‐gated Ca2+ channels, whereas stimulation of NMDA receptors caused Ca2+ influx through NMDA‐gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE‐binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down‐regulation of PKC inhibited the increase in TRE‐binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca2+‐signaling pathways for the increase in TRE‐binding activity coupled with the activation of NMDA‐ and non‐NMDA receptors.

Additive induction of Egr-1 (zif/268) mRNA expression in neuroblastoma × glioma hybrid NG108-15 cells via cholinergic muscarinic, α2-adrenergic, and bradykinin receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr‐1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr‐1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α2‐adrenergic receptors, respectively. At their saturation levels for Egr‐1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr‐1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr‐1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr‐1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr‐1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr‐1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr‐1 induction, depending on the times of stimulation and the combinations of receptors stimulated.

Preventive effects of bifemelane hydrochloride on decreased levels of muscarinic acetylcholine receptor and its mRNA in a rat model of chronic cerebral hypoperfusion

Changes in muscarinic acetylcholine receptor (mACh-R) binding and muscarinic cholinergic m1 receptor (m1-R) mRNA levels were determined in a rat model of cerebral hypoperfusion in which hypoperfusion was induced by permanent bilateral occlusion of the common carotid arteries. After 6 weeks of hypoperfusion, mACh-R binding activity was significantly reduced in the frontal cortex (79.0 percent, P <0.01), striatum (74.2 percent, P < 0.01) and hippocampus (78.6 percent, P < 0.01), and the m1-R mRNA levels in the frontal cortex (86.6 percent, P < 0.05) and striatum (89.4 percent, P < 0.05) compared with sham-operated control. Repeated administration of bifemelane hydrochloride (15 mg/kg/day, p.o., once a day from the day of operation for 6 weeks) prevented the hypoperfusion-induced loss of mACh-R binding and m1-R mRNA levels above described. Since the central cholinergic systems play an important role in learning and memory, these findings suggest that bifemelane hydrochloride is useful to treat and/or prevent vascular dementia which is closely related to cerebral hypoperfusion.