Kumiko Ishige

FLAVONOIDS PROTECT NEURONAL CELLS FROM OXIDATIVE STRESS BY THREE DISTINCT MECHANISMS

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimers and Parkinsons. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.

Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome.

S-allyl-l-cysteine selectively protects cultured rat hippocampal neurons from amyloid β-protein- and tunicamycin-induced neuronal death

S-allyl-L-cysteine (SAC), one of the organosulfur compounds found in aged garlic extract, has been shown to possess various biological effects including neurotrophic activity. In our previous experiments, we found that SAC could protect against amyloid beta-protein (Abeta)- and tunicamycin-induced cell death in differentiated PC12 cells. In the study described here, we characterized the neuronal death induced by Abeta, 4-hydroxynonenal (HNE), tunicamycin, and trophic factor deprivation, and investigated whether and how SAC could prevent this in cultured rat hippocampal neurons. Treatment with SAC protected these cells against Abeta- and tunicamycin-induced neuronal death, which is mediated predominantly through caspase-12-dependent pathway in a concentration-dependent manner. In contrast, it afforded no protection against HNE- and trophic factor-deprivation-induced cell death, which has been shown to be mediated by caspase-3-dependent pathway. SAC also attenuated the Abeta-induced increase of intracellular reactive oxygen species in hippocampal neurons. SAC had no effect on Abeta-induced cell death in cultured cerebellar granule neurons, which was prevented by a caspase-3 inhibitor. These results suggest that SAC could protect against the neuronal cell death that is triggered by ER dysfunction in the hippocampus, and that it has no effect on neuronal cell death that is dependent upon the caspase-3 mediated pathway.

Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP+-affected mesencephalic dopaminergic cells

Summary.Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP+). Ginsenoside Rb1 (at 10 µM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP+ (at 1 µM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH+ cells. Both compounds however could not prevent the cell loss caused by MPP+. Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.

Protective effects on neuronal cells of mouse afforded by ebselen against oxidative stress at multiple steps

Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) mimics the activity of glutathione peroxidase [Biochem. Pharmacol. 33 (1984) 3235], acts as a substrate for thioredoxin reductase [Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 8579]. The present study focused on the cellular mechanism of its action against oxidative stress by using HT22 cells, a mouse neuroblastoma of hippocampal origin. Ebselen protected HT22 cells against death induced by glutamate and hydrogen peroxide but not against that by tumor necrosis factor alpha. Oxidative glutamate toxicity is initiated by depletion of total glutathione, and ebselen inhibited the decrease in glutathione and increased its basal level. Although glutamate increased intracellular levels of reactive oxygen species (ROS), ebselen suppressed their increase. Ebselen reduced the basal levels of ROS when it was applied in control cells. Ebselen also removed ROS from cells that had accumulated a level of them. The compound had a significant trolox equivalent activity concentration value in a cell-free system, suggesting that it has a direct ROS-scavenging capacity. Finally, ebselen-induced heme oxygenase-1 (HO-1) protein. These results indicate that ebselen protects neuronal cells against the oxidative stress at multiple steps, including an increase in glutathione, a ROS-scavenging activity and the induction of HO-1 protein.

γ-hydroxybutyric acid increases intracellular Ca2+ concentration and nuclear cyclic AMP-responsive element- and activator protein 1 DNA-binding activities through GABAB receptor in cultured cerebellar granule cells

Abstract: In primary cultures of mouse cerebellar granule cells, a brief stimulation by γ‐hydroxybutyric acid (GHB, 0.1–3 mM) significantly increased the intracellular Ca2+ concentration ([Ca2+]i) in a concentration‐dependent manner. In addition, gel mobility assay showed that exposure of the cells to GHB also increased nuclear DNA‐binding activity specific for the cyclic AMP‐responsive element (CRE) and activator protein 1 (AP‐1) transcriptional element in a concentration‐dependent manner. The concentration range of GHB that increased the DNA‐binding activity was essentially the same as the concentration range that elicited the increase in [Ca2+]i. The GHB‐induced increases in [Ca2+]i and nuclear DNA‐binding activity were antagonized by specific GABAB antagonists such as p‐[3‐aminopropyl]‐p‐diethoxymethylphosphinic acid (CGP 35 348) and 3‐N‐[1‐(S)‐(3,4‐dichlorobenzyl)ethanol‐2‐(S)‐hydroxy‐P‐benzylphosphinic acid (CGP 55 845). In addition, the GHB‐induced increase in [Ca2+]i was abolished by pretreatment of the cells with islet‐activating protein. Furthermore, treatment of the cells with 1,2‐bis(2′‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (BAPTA‐AM) and thapsigargin blocked the GHB‐induced increase in nuclear DNA‐binding activity. GHB inhibited [3H]baclofen binding to cultured cerebellar granule cells and mouse cerebellar membranes. These results suggest that stimulation of GABAB receptors by GHB activates intracellular Ca2+ stores and that the increased [Ca2+]i resulting from release of stored Ca2+ plays an important role in increasing the CRE‐ and AP‐1 DNA‐binding activities in cultured cerebellar granule cells.

Ammonium tetrathiomolybdate delays onset, prolongs survival, and slows progression of disease in a mouse model for amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) cause a form of familial amyotrophic lateral sclerosis (ALS). The pathogenesis of familial ALS may be associated with aberrant copper chemistry through a cysteine residue in mutant SOD1. Ammonium tetrathiomolybdate (TTM) is a copper-chelating drug that is capable of removing a copper ion from copper-thiolate clusters, such as SOD1. We found that TTM exerted therapeutic benefits in a mouse model of familial ALS (SOD1(G93A)). TTM treatment significantly delayed disease onset, slowed disease progression and prolonged survival by approximately 20%, 42% and 25%, respectively. TTM also effectively depressed the spinal copper ion level and inhibited lipid peroxidation, with a significant suppression of SOD1 enzymatic activity in SOD1(G93A). These results support the hypothesis that aberrant copper chemistry through a cysteine residue plays a critical role in mutant SOD1 toxicity and that TTM may be a promising therapy for familial ALS with SOD1 mutants.

N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death

4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity accompanied by multiple events. To clarify mechanisms of neuroprotective compounds on HNE-induced toxicity, the protective effects of N-acetylcysteine (NAC), alpha-tocopherol (TOC), ebselen and S-allyl-L-cysteine (SAC) were compared in cerebellar granule neurons. The decrease in MTT reduction induced by HNE was significantly suppressed by pretreatment of the neurons with 1000 microM NAC or 10 and 100 microM TOC; however, lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence studies revealed that neuronal death was suppressed by NAC but not by TOC. Treatment of these neurons with HNE resulted in a drastic reduction of mitochondrial membrane potential, and this reduction was also prevented by NAC but not by TOC. Ebselen and SAC, a garlic compound, were unable to protect these neurons against HNE-induced toxicity. Pretreatment with NAC also prevented HNE-induced depletion of intracellular glutathione (GSH) levels in these neurons. These results suggest that NAC, but not other antioxidants such as TOC, SAC and ebselen, exerts significant protective effects against HNE-induced neuronal death in cerebellar granule neurons, and that this neuroprotective effect is due, at least in part, to preservation of mitochondrial membrane potential and intracellular GSH levels.

Neurotoxicity induced by amyloid β-peptide and ibotenic acid in organotypic hippocampal cultures: protection by S-allyl-l-cysteine, a garlic compound

We have assessed amyloid-beta (Abeta)-induced neurotoxicity, with and without added ibotenic acid (IBO), a potent N-methyl-D-aspartate (NMDA) agonist, in an organotypic hippocampal slice culture (OHC). In the OHC, there was little neurotoxicity after treatment with Abeta(25-35) (25 or 50 microM) alone for 48 h. However, with IBO alone neuronal death was observed in the pyramidal cell layer at low concentrations, and there was dramatic neuronal death at concentrations of 65 microM or more. When Abeta was combined with IBO (Abeta+IBO) there was more intense cell death than with IBO alone. S-Allyl-L-cysteine (SAC), one of the organosulfur compounds having a thioallyl group in aged garlic extract, was shown to protect the hippocampal neurons in the CA3 area and the dentate gyrus (DG) from the cell death induced by Abeta+IBO with no change in the CA1 area. Although L-glutamate (500 microM) potentiated the degree of IBO-induced neuronal death, it attenuated the Abeta+IBO-induced neuronal death in both the CA3 area and the DG with no obvious effect on the CA1 area. These results suggest that Abeta+IBO induces extensive neuronal death, and that SAC and L-glutamate protect cells from death in specific areas of the hippocampus. In addition, inhibition using a pan-caspase inhibitor, z-VAD-fmk, only provided partial protection from Abeta+IBO-induced toxicity for the neurons in the CA3 area. These results suggest that multiple mechanisms may be involved in Abeta+IBO-induced neuronal death in the OHC.

Comparative study of survival signal withdrawal- and 4-hydroxynonenal-induced cell death in cerebellar granule cells

The lipid peroxidation product, 4-hydroxynonenal (HNE), has been shown to induce apoptosis in PC12 cells and hippocampal neurons. We compared the degree of cell death induced by survival signal withdrawal (K+ and serum deprivation) with that induced by HNE, and investigated whether agents that block survival signal withdrawal-induced apoptosis could also prevent HNE-induced cell death in cultured cerebellar granule cells. Cell death induced by K+ and serum deprivation was inhibited by cycloheximide, a CPP 32-like protease inhibitor (Ac-DEVD-CHO) and a pituitary adenylate cyclase-activating polypeptide (PACAP)-38. In addition, nuclear cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities were increased 2 h after K+ and serum withdrawal, and these increases were inhibited by cycloheximide, Ac-DEVD-CHO and PACAP 38. Although these agents also blocked HNE-induced cell death, consistent with their efficacy in preventing survival signal withdrawal-induced cells death, CRE and AP-1 DNA-binding activities were decreased in a time-dependent manner during HNE-induced cell death. These results suggest that mechanistic differences exist between apoptosis induced by HNE and that induced by withdrawal of survival signals in cerebellar granule neurons.