Yutaka Sagara

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.

α-Synuclein Promotes Mitochondrial Deficit and Oxidative Stress

Abnormal accumulation of the presynaptic protein α-synuclein has recently been implicated in the pathogenesis of Alzheimer’s and Parkinson’s diseases. Because neurodegeneration in these conditions might be associated with mitochondrial dysfunction and oxidative stress, the effects of α-synuclein were investigated in a hypothalamic neuronal cell line (GT1-7). α-Synuclein overexpression in these cells resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations accompanied by increased levels of free radicals and decreased secretion of gonadotropin-releasing hormone. These alterations were ameliorated by pretreatment with anti-oxidants such as vitamin E. Taken together these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that may result in oxidative stress and, eventually, cell death.

β-Amyloid peptides enhance α-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease

Alzheimers disease and Parkinsons disease are associated with the cerebral accumulation of β-amyloid and α-synuclein, respectively. Some patients have clinical and pathological features of both diseases, raising the possibility of overlapping pathogenetic pathways. We generated transgenic (tg) mice with neuronal expression of human β-amyloid peptides, α-synuclein, or both. The functional and morphological alterations in doubly tg mice resembled the Lewy-body variant of Alzheimers disease. These mice had severe deficits in learning and memory, developed motor deficits before α-synuclein singly tg mice, and showed prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals. They also had more α-synuclein-immunoreactive neuronal inclusions than α-synuclein singly tg mice. Ultrastructurally, some of these inclusions were fibrillar in doubly tg mice, whereas all inclusions were amorphous in α-synuclein singly tg mice. β-Amyloid peptides promoted aggregation of α-synuclein in a cell-free system and intraneuronal accumulation of α-synuclein in cell culture. β-Amyloid peptides may contribute to the development of Lewy-body diseases by promoting the aggregation of α-synuclein and exacerbating α-synuclein-dependent neuronal pathologies. Therefore, treatments that block the production or accumulation of β-amyloid peptides could benefit a broader spectrum of disorders than previously anticipated.

Induction of Reactive Oxygen Species in Neurons by Haloperidol

Abstract: Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT‐22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT‐22 cells are equally sensitive to HP with an IC50 of 35 µM in the primary neurons and 45 µM in HT‐22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as β‐estradiol and vitamin E, lowers the level of ROS and protects diol and vitamin E, lowers the level of ROS and protects HT‐22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca2+, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimers disease, and Parkinsons disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.

Fibroblast Growth Factor 1 Regulates Signaling via the Glycogen Synthase Kinase-3β Pathway IMPLICATIONS FOR NEUROPROTECTION

We hypothesize that in neurodegenerative disorders such as Alzheimers disease and human immunodeficiency virus encephalitis the neuroprotective activity of fibroblast growth factor 1 (FGF1) against several neurotoxic agents might involve regulation of glycogen synthase kinase-3β (GSK3β), a pathway important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a time-dependent inactivation of GSK3β by phosphorylation at serine 9. Blocking FGF1 receptors with heparinase reduced this effect. The effects of FGF1 on GSK3β were dependent on phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) because inhibitors of this pathway or infection with dominant negative Akt adenovirus blocked inactivation. Furthermore, treatment of neuronal cells with FGF1 resulted in ERK-independent Akt phosphorylation and β-catenin translocation into the nucleus. On the other hand, infection with wild-type GSK3β recombinant adenovirus-associated virus increased activity of GSK3β and cell death, both of which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity was dependent on GSK3β inactivation by the PI3K-Akt but was independent of ERK. Taken together these results suggest that neuroprotective effects of FGF1 might involve inactivation of GSK3β by a pathway involving activation of the PI3K-Akt cascades.

Vitamin E supplementation prevents spatial learning deficits and dendritic alterations in aged apolipoproteinE‐deficient mice

Recent studies have suggested that altered function of apolipoprotein E might lead to Alzheimers disease via oxidative stress. In this context, the objective of this study was to determine if antioxidative treatment with vitamin E was neuroprotective in apolipoprotein E‐deficient mice. For this purpose, 1‐month‐old control and apolipoprotein E‐deficient mice received dietary vitamin E for 12 months. We showed that, compared to apolipoprotein E‐deficient mice who received a regular diet, mice treated with vitamin E displayed a significantly improved behavioural performance in the Morris water maze. This improved performance was associated with preservation of the dendritic structure in vitamin E‐treated apolipoprotein E‐deficient mice. In addition, whilst untreated apolipoprotein E‐deficient mice displayed increased levels of lipid peroxidation and glutathione, vitamin E‐treated mice showed near normal levels of both lipid peroxidation and glutathione. These results support the contention that vitamin E prevents the age‐related neurodegenerative alterations in apolipoprotein E‐deficient mice.

Propofol Hemisuccinate Protects Neuronal Cells from Oxidative Injury

Abstract : Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood—brain barrier (BBB). Propofol (2,6‐diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water‐soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non‐receptor‐mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT‐22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT‐22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT‐22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested α‐tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT‐22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water‐soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.

Neurotoxic effects of apolipoprotein E4 are mediated via dysregulation of calcium homeostasis

The association of the E4 allele of apolipoprotein E (apoE4) as a genetic risk factor for Alzheimers disease (AD) has been well established. Although recent studies in neuronal cell lines and transgenic mice have shown that apoE4 promotes neurodegeneration, the mechanisms through which apoE4 impairs neuronal viability are not completely understood. In this context, the main objective of the present study was to determine whether the neurotoxic effects of apoE4 are mediated by an alteration in calcium homeostasis. For this purpose, effects of recombinant apoE3 and apoE4 on cell viability and intracellular calcium levels were analyzed in a murine hippocampal cell line (HT22) and in primary rat cortical neurons, in the presence or absence of calcium inhibitors. Under basal conditions, apoE4‐treated cells displayed increased levels of cytosolic calcium associated with cell death in a dose‐dependent manner. Furthermore, apoE4 treatment potentiated the rise in cytosolic calcium and cell death following the administration of a calcium ionophore. The effects of apoE4 on cell viability and calcium homeostasis were inhibited by calcium chelators or by blocking calcium channels, but not by inhibitors of intracellular calcium reserves. Taken together, these results indicate that the neurotoxic effects of apoE4 are dependent on extracellular calcium influx via calcium channels.

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.