[Show abstract][Hide abstract] ABSTRACT: Oxidative stress and the ubiquitin–proteasome system play a key role in the pathogenesis of Parkinson disease. Although the herbicide paraquat is an environmental factor that is involved in the etiology of Parkinson disease, the role of 26S proteasome in paraquat toxicity remains to be determined. Using PC12 cells overexpressing a fluorescent protein fused to the proteasome degradation signal, we report here that paraquat yielded an inhibitory effect on 26S proteasome activity without an obvious decline in 20S proteasome activity. Relative low concentrations of proteasome inhibitors caused the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is targeted to the ubiquitin–proteasome system, and activated the antioxidant response element (ARE)-dependent transcription. Paraquat also upregulated the protein level of Nrf2 without increased expression of Nrf2 mRNA, and activated the Nrf2–ARE pathway. Consequently, paraquat induced expression of Nrf2-dependent ARE-driven genes, such as γ-glutamylcysteine synthetase, catalase, and hemeoxygenase-1. Knockdown of Nrf2 or inhibition of γ-glutamylcysteine synthetase and catalase exacerbated paraquat-induced toxicity, whereas suppression of hemeoxygenase-1 did not. These data indicate that the compensatory activation of the Nrf2–ARE pathway via inhibition of 26S proteasome serves as part of a cellular defense mechanism to protect against paraquat toxicity.
Preview · Article · Oct 2015 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that serofendic acid, a lipophilic extract of fetal calf serum, protects against oxidative stress in primary culture of neonatal rat cardiomyocytes. However, the effect of serofendic acid on myocardial ischemia-reperfusion injury in vivo is yet to be determined. In the present study, we investigated the effect of intravenous administration of serofendic acid on ischemia-reperfusion injury induced by transient occlusion of the left coronary artery in rats. The rat heart was subjected to 25-min ischemia followed by 2-h reperfusion. Bolus intravenous administration of serofendic acid (1 - 10 mg/kg) given twice reduced the infarct volume in a dose-dependent manner. The protective effect of serofendic acid was abolished by pretreatment with 5-hydroxydecanoate, a blocker of mitochondrial ATP-sensitive potassium channels. For further testing of the protective effect of serofendic acid at the subcellular level, we monitored mitochondrial membrane potential (MMP) in individual cells using real-time two-photon imaging of Langendorff-perfused rat heart. A 25-min no-flow ischemia, followed by reperfusion caused progressive MMP loss. Serofendic acid significantly reduced the number of cells undergoing MMP loss. These results suggest that serofendic acid protected cardiac myocytes against myocardial ischemia-reperfusion injury by preserving the functional integrity of mitochondria.
No preview · Article · Nov 2014 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Introduction. Our previous data showed that treatment of nicotine or donepezil prevented glutamate-induced cytotoxicity via nicotinic receptors (nAChRs) using primary culture of rat cortical neurons. The present study was performed to investigate the detailed mechanisms of nAChRs-mediated neuroprotection, especially involvement of glycogen synthase kinase-3β(GSK3 β ) as a downstream of PI3K-Akt pathway.
Methods. Neuronal death was determined by LDH release assay in rat primary culture of cerebral cortex. Phosphorylation of GSK3β and the expression level of β-catenin were measured by western blot analysis.
Results and Conclusion. Donepezil induced the Ser9-phosphorylation of GSK3β in rat cultured cortical neurons. LY294002, an inhibitor of PI3K, prevented that phosphorylation of GSK3β. Glutamate induced the Tyr216-phosphorylation of GSK3β. Donepezil prevented glutamate-induced phosphorylation of Tyr216. Bcl-2 was upregulated by donepezil treatment, but not by SB216763, an inhibitor of GSK3β treatment. On the other hand, the expression level of β-catenin was increased by both donepezil and SB216763. These results suggested that inactivation of GSK3β as the downstream signaling of PI3K-Akt pathway play a crucial role in the nAChR-mediated neuroprotection.
[Show abstract][Hide abstract] ABSTRACT: The herbicide paraquat is an environmental factor that may be involved in the etiology of Parkinson's disease (PD). Systemic
exposure of mice to paraquat causes a selective loss of dopaminergic neurons in the substantia nigra pars compacta, although
paraquat is not selectively incorporated in dopaminergic neurons. Here, we report a contribution of endogenous dopamine to
paraquat-induced dopaminergic cell death. Exposure of PC12 cells to paraquat (50μM) caused delayed toxicity from 36 h onward.
A decline in intracellular dopamine content achieved by inhibiting tyrosine hydroxylase (TH), an enzyme for dopamine synthesis,
conferred resistance to paraquat toxicity on dopaminergic cells. Paraquat increased the levels of cytosolic and vesicular
dopamine, accompanied by transiently increased TH activity. Quinone derived from cytosolic dopamine conjugates with cysteine
residues in functional proteins to form quinoproteins. Formation of quinoprotein was transiently increased early during exposure
to paraquat. Furthermore, pretreatment with ascorbic acid, which suppressed the elevations of intracellular dopamine and quinoprotein,
almost completely prevented paraquat toxicity. These results suggest that the elevation of cytosolic dopamine induced by paraquat
participates in the vulnerability of dopaminergic cells to delayed toxicity through the formation of quinoproteins.
[Show abstract][Hide abstract] ABSTRACT: Curcumin, a polyphenolic compound has several pharmacological activities, such as anticancer, anti-inflammatory and antioxidant effects. However, curcumin shows poor oral bioavailability. The purpose of this study was to investigate the protective effects of highly bioavailable curcumin, Theracurmin(®), and curcumin, against sodium nitroprusside (SNP)-induced oxidative damage in mice brain. Intrastriatal microinjection of Theracurmin(®) or curcumin with SNP significantly protected against SNP-induced brain damage and motor dysfunction. Oral administration of Theracurmin(®) (1 and 3 g kg(-1), containing 100 and 300 mg kg(-1) curcumin, respectively) significantly protected against SNP-induced brain damage and motor dysfunction. However, oral administration of 300 mg kg(-1) curcumin did not protect against motor dysfunction induced by SNP. These results suggest that curcumin and Theracurmin(®) have protective effects against SNP-induced oxidative damage. Moreover, oral administration of Theracurmin(®), had more potency in protecting against brain damage, suggesting a higher bioavailability of Theracurmin(®) following oral administration.
[Show abstract][Hide abstract] ABSTRACT: Axonal degeneration of dopaminergic neurons is one of the pathological features in the early stages of Parkinson disease. Promotion of axonal outgrowth of the remaining dopaminergic neurons leads to the recovery of the nigrostriatal pathway. Staurosporine (STS), a wide-spectrum kinase inhibitor, induces neurite outgrowth in various cell types, although its mechanism of action remains elusive. In this study, we analyzed which protein kinase is involved in STS-induced neurite outgrowth. We have previously established the method to measure the length of dopaminergic neurites that extend from a mesencephalic cell region, which is formed on a coverslip by an isolation wall. By means of this method, we clarified that STS treatment causes dopaminergic axonal outgrowth in mesencephalic primary cultures. Among the specific protein kinase inhibitors we tested, compound C (C.C), an AMP-activated protein kinase (AMPK) inhibitor, promoted dopaminergic neurite outgrowth. STS as well as C.C elevated the phosphorylation level of 70-kDa ribosomal protein S6 kinase, a downstream target of mammalian target of rapamycin (mTOR) signaling pathway. The STS- and C.C-induced dopaminergic neurite outgrowth was suppressed by rapamycin, an mTOR inhibitor. Furthermore, the application of C.C rescued 1-methyl-4-phenylpyridinium ion (MPP(+))-induced dopaminergic neurite degeneration. These results suggest that STS induces dopaminergic axonal outgrowth through mTOR signaling pathway activation as a consequence of AMPK inhibition.
No preview · Article · Sep 2013 · Neuropharmacology
[Show abstract][Hide abstract] ABSTRACT: Serofendic acid is a low-molecular-weight compound extracted from fetal calf serum. We previously reported that intracerebroventricular administration of serofendic acid prevents cerebral ischemia-reperfusion injury. However, the effect of peripheral administration of serofendic acid on cerebral ischemia-reperfusion injury has not been examined. In the present study, we investigated the effect of intravenous administration of serofendic acid against cerebral ischemia-reperfusion injury using transient middle cerebral artery occlusion model rats. Serofendic acid (10mg/kg) administrated three times, including 30 minutes before the onset of ischemia, just after the onset of ischemia and just before reperfusion reduced the infarct volume and improved the neurological dysfunction induced by ischemia-reperfusion without affecting regional cerebral blood flow or physiological parameters. However, there were no protective effects when serofendic acid (30mg/kg) was only administered once at 30min before the onset of ischemia, just after the onset of ischemia, or just before reperfusion. Our results reveal the importance of maintaining the blood concentration of serofendic acid for preventing cerebral ischemia-reperfusion injury.
[Show abstract][Hide abstract] ABSTRACT: Curcumin, a polyphenolic compound extracted from Curcuma longa, has several pharmacological activities such as anticancer, anti-inflammatory, and antioxidant effects. The purpose of this study was to investigate the protective effects of curcumin and THERACURMIN, a highly bioavailable curcumin, against sodium nitroprusside (SNP)-induced oxidative damage in primary striatal cell culture. THERACURMIN as well as curcumin significantly prevented SNP-induced cytotoxicity. To elucidate the cytoprotective effects of curcumin and THERACURMIN, we measured the intracellular glutathione level in striatal cells. Curcumin and THERACURMIN significantly elevated the glutathione level, which was decreased by treatment with SNP. Moreover, curcumin showed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability. Finally, a ferrozine assay showed that curcumin (10-100 µg/mL) has potent Fe(2+)-chelating ability. These results suggest that curcumin and THERACURMIN exert potent protective effects against SNP-induced cytotoxicity by free radical-scavenging and iron-chelating activities.
No preview · Article · Aug 2013 · Biological & Pharmaceutical Bulletin
[Show abstract][Hide abstract] ABSTRACT: The 42-mer amyloid β-protein (Aβ42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer's disease (AD). We previously identified the toxic conformer of Aβ42 with a turn at positions 22-23 ("toxic" turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25-26. G25P-Aβ42 and E22V-Aβ42 are non-toxic mutants that disfavor the "toxic" turn. Here we hypothesize that these non-toxic mutants of Aβ42 could suppress the neurotoxicity of Aβ42, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aβ42 and E22V-Aβ42 suppressed the neurotoxicity and aggregation of Aβ42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aβ42 was also reduced significantly by the treatment of 11A1, but not of Aβ-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aβ oligomers contain parallel β-sheet, the present results suggest that the non-toxic mutants of Aβ42 without the "toxic" turn could prevent the propagation process of the toxic conformer of Aβ42 resulting in suppression of the formation of the toxic oligomers. This may be a promising strategy for AD therapeutics.
Full-text · Article · Jun 2013 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Accumulating lines of evidence showed that luteolin, a polyphenolic compound, has potent neuroprotective effects. The purpose of this study was to examine whether luteolin can protect against sodium nitroprusside (SNP)-induced oxidative damage in mouse brain. Intrastriatal co-injection of luteolin (3 - 30 nmol) with SNP (10 nmol) dose-dependently protected against brain damage and motor dysfunction. Oral administrations of luteolin (600 - 1200 mg/kg) dose-dependently protected against brain damage and motor dysfunction induced by striatal injection of SNP. Furthermore, luteolin (30 - 100 μM) concentration dependently protected against Fe(2+)-induced lipid peroxidation in mouse brain homogenate. Luteolin (1 - 100 μg/ml) showed potent DPPH radical scavenging ability, when compared with ascorbic acid and glutathione. Finally, a ferrozine assay showed that luteolin (30 - 100 μg/ml) has Fe(2+)-chelating ability, but this was weaker than that of ethylenediaminetetraacetic acid. These results suggest that intrastriatal or oral administration of luteolin protected mice brain from SNP-induced oxidative damage by scavenging and chelating effects.
No preview · Article · May 2013 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Sodium nitroprusside (SNP) is widely used as a potent vasodilator and a nitric oxide (NO) donor, whereas the cytotoxicity of SNP has been well documented. SNP releases several potentially toxic products such as cyanide anion, NO, and iron. We investigated the mechanisms of cell death and motor dysfunction induced by microinjection of SNP in mice to establish a brain oxidative stress model and then examined the anti-oxidant activity of glutathione. Intrastriatal microinjection of SNP (1 - 10 nmol) induced brain damage and motor dysfunction in a dose-dependent manner when the effects were evaluated with behavioral tests and TTC staining. NOC-18 (10 nmol), another NO donor, and KCN (10 nmol) did not cause motor dysfunction. However, FeCl(2) (10 nmol) caused motor dysfunction. In addition, simultaneous injection of SNP and deferoxamine (10 nmol), an iron-chelating agent, prevented SNP-induced brain damage and motor dysfunction, suggesting a role of iron-related radicals in SNP-toxicity. Moreover, reduced glutathione (1 - 10 nmol), a natural anti-oxidant substance, dose-dependently prevented motor dysfunction induced by SNP-toxicity. Finally, deferoxamine and glutathione (10 nmol) significantly protected against brain damage and motor dysfunction induced by FeCl(2) toxicity. These results suggest that cell death induced by injection of SNP is caused by iron-related radical reactions, but not by NO and cyanide anion.
No preview · Article · Sep 2012 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: The 42-mer amyloid β-protein (Aβ42) aggregates to form soluble oligomers that cause memory loss and synaptotoxicity in Alzheimer's disease (AD). Oxidative stress is closely related to the pathogenesis of AD. We previously identified the toxic conformer of Aβ42 with a turn at positions 22 and 23 ("toxic turn") by solid-state NMR and demonstrated that a monoclonal antibody (11A1) against the toxic turn in Aβ42 mainly detected the oligomer in the brains of AD patients. Our recent study suggested that oxidative stress is a key factor of the oligomerization and cognitive impairment induced by Aβ overproduction in vivo. However, the involvement of the toxic conformer in Aβ42-induced oxidative damage remains unclear. To investigate this mechanism, we examined the levels of intracellular reactive oxygen species (ROS) and neurotoxicity in rat primary neurons using E22P-Aβ42, a mutant that induces a turn at positions 22 and 23, and E22V-Aβ42, a turn-preventing mutant. E22P-Aβ42, but not E22V-Aβ42, induced greater ROS production than Wt-Aβ42 in addition to potent neurotoxicity. Interestingly, the formation of the toxic conformer in both E22P-Aβ42 and Wt-Aβ42 probed by the 11A1 antibody preceded Aβ42-induced neurotoxicity. Trolox (a radical scavenger) and Congo red (an aggregation inhibitor) significantly prevented the neurotoxicity and intracellular ROS induced by E22P-Aβ42 and Wt-Aβ42, respectively. These results suggest that Aβ42-mediated toxicity is caused by the turn that favors toxic oligomers, which increase generation of ROS.
No preview · Article · Sep 2012 · ACS Chemical Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Rotenone, a mitochondrial complex I inhibitor, has been used to generate animal and cell culture models of Parkinson's disease. Recent studies suggest that microtubule destabilization causes selective dopaminergic neuronal loss. In this study, we investigated glycogen synthase kinase-3β (GSK3β) involvement in rotenone-induced microtubule destabilization. Rotenone-induced cytotoxicity in SH-SY5Y cells was attenuated by the GSK3β inhibitor SB216763. Tau, a microtubule-associated protein and substrate for GSK3β, has been implicated in the pathogenesis of tauopathies such as Alzheimer's disease. Rotenone induced an increase in phosphorylated tau, the effect of which was attenuated by concomitant treatment with SB216763. Rotenone treatment also decreased tau expression in the microtubule fraction and increased tau expression in the cytosol fraction. These effects were suppressed by SB216763, which suggests that rotenone reduces the capacity of tau to bind microtubules. Rotenone treatment increased the amount of free tubulin and reduced the amount of polymerized tubulin, indicating that rotenone destabilizes microtubules. Rotenone-induced microtubule destabilization was suppressed by SB216763 and taxol, a microtubule stabilizer. Taxol prevented rotenone-induced cytotoxicity and morphological changes. Taken together, these results suggest that rotenone-induced cytotoxicity is mediated by microtubule destabilization via GSK3β activation, and that microtubule destabilization is caused by reduction in the binding capacity of tau to microtubules, which is a result of tau phosphorylation via GSK3β activation.
No preview · Article · Aug 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.
No preview · Article · Jun 2012 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids are stress hormones released from the adrenal cortex and their concentration is controlled by the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of glucocorticoids on the number of astrocytes and glucocorticoid receptor (GR) expression in vitro and in vivo. Proliferation of cultured astrocytes was reduced following treatment with corticosterone and dexamethasone for 72 h. Corticosterone and dexamethasone also reduced GR expression in astrocytes. RU486, a GR antagonist, inhibited the reduction in both astrocyte proliferation and GR expression. Furthermore, GR knockdown by siRNA inhibited astrocyte proliferation. We also examined the effect of excessive glucocorticoid release on GR expression and the number of astrocytes in vivo by administering adrenocorticotropic hormone to rats for 14 days. GR expression was reduced in the prefrontal cortex and hippocampus and the number of astrocytes was reduced in the frontal cortex. Overall, our results suggest that glucocorticoids decrease the number of astrocytes by reducing GR expression.
No preview · Article · Apr 2012 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Hydrogen sulfide (H(2)S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H(2)S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H(2)S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.
Preview · Article · Nov 2011 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: FE65 is reported to act as an adaptor protein with several protein-interaction domains, including one WW domain and two phosphotyrosine interaction/binding domains. Through these binding domains, FE65 was considered to recruit various binding partners together to form functional complexes in a certain cellular compartment. In this study, we demonstrated that Rac1, a member of the Rho family GTPases, bound with FE65. We also elucidated that Rac1 inhibitor significantly suppressed FE65 expression, and Rac1 small interfering RNA transduction significantly decreased FE65 expression. FE65 small interfering RNA, however, did not influence Rac1 expression and its activity. Taken together, our results reveal that Rac1 interacts with FE65, and Rac1 activity regulates FE65 expression.