Pregnenolone Protects Mouse Hippocampal (HT-22) Cells against Glutamate and Amyloid Beta Protein Toxicity

In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 M amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 M amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.     

Tamoxifen protects clonal mouse hippocampal (HT-22) cells against neurotoxins-induced cell death

In the present work using an established clonal mouse hippocampal (HT-22) cell line, we have examined whether the estrogen antagonist tamoxifen antagonizes the observed neuroprotective effects of estrogen against glutamate and amyloid beta protein neurotoxicity. Results obtained suggest that like estrogen, tamoxifen protects HT-22 cells against both 5mM glutamate and 2 microM amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was obtained at 500 nM tamoxifen. Tamoxifen was found to offer more potent protection at this dose against amyloid beta protein induced neurotoxicity when compared with glutamate neurotoxicity. We were unable to detect either estrogen receptor (ER)--ER alpha or ER beta presence in HT-22 cells using western blot technique. However, amyloid beta protein treatment significantly increases total glucocorticoid receptors (GRs) as determined by western blot technique, while prior treatment with estrogen or tamoxifen followed by amyloid beta protein resulted in the reduction of total GRs to the levels comparable to that observed for the control untreated cells. In addition, using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 2 microM amyloid beta protein resulted in enhanced nuclear localization of GRs in HT-22 cells as compared to control untreated cells or 500 nM tamoxifen alone treated cells. Interestingly, 500 nM tamoxifen treatments for 24h, followed by 20 h treatment with 2 microM amyloid beta protein resulted in dramatic reduction in GRs nuclear localization. In conclusion, tamoxifen (i) protects HT-22 cells against amyloid beta protein neurotoxicity and (ii) neuroprotective effect is independent of ERs.     

Dehydroepiandrosterone protects hippocampal neurons against neurotoxin-induced cell death: mechanism of action

Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels.     

Heat shock preconditioning and pretreatment with glucocorticoid antagonist RU 486 protect rat myogenic cells H9c2 against glutamate-induced cell death

We have observed that the treatment of rat-heart derived H9c2 myoblasts for 20 h with the excitatory amino acid glutamate resulted in cell death in a dose dependent manner as determined by LDH release. The optimum cardiotoxicity was seen at 25 mM glutamate. Preconditioning with either sublethal heat shock (42°C for 30 min) or pretreatment with 500 nM of the glucocorticoid antagonist RU 486 for 24 h almost completely protected H9c2 cells against subsequent 20 h treatment with 25 mM lethal glutamate. In addition, we have observed that glutamate treatment resulted in intense nuclear localization of glucocorticoid receptors (GR) in H9c2 cells as judged by the confocal immunofluorescence microscopy. Furthermore, pretreatment with either heat shock or RU 486 followed by glutamate treatment resulted in dramatic decrease in GR nuclear localization which was almost comparable to that observed with control untreated cells. In conclusion, we have shown for the first time using H9c2 cells that (i) protection from glutamate cardiotoxicity occurs with prior treatment with sub lethal heat shock or RU 486 and (ii) these measures down regulate the intense nuclear localization of GR induced by glutamate. The block to GR nuclear localization is likely to be involved in cardioprotective effects offered against glutamate toxicity by pretreatment with heat shock or RU 486.     

Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.     

Role of nonfeminizing estrogen analogues in neuroprotection of rat retinal ganglion cells against glutamate-induced cytotoxicity

Glaucoma is a family of eye disorders whose ultimate cause of vision loss is apoptosis of retinal ganglion cells. Although several etiologies of glaucoma exist, oxidative stress is thought to be a key mechanism by which ganglion cells die. From this perspective, the work presented here was designed to examine the efficacy of 17beta-estradiol and three synthetic estrogen analogues (ZYC-1, ZYC-3, ZYC-10) as retinal ganglion cell neuroprotectants. Compound ZYC-1 and its enantiomer ZYC-10, containing an additional double bond in the steroid C ring of 17beta-estradiol, had similar (ZYC-1) or modestly reduced (ZYC-10) affinity for estrogen receptors compared to the parent estrogen. In the case of ZYC-3, the addition of an adamantyl group to the C2 position of the A ring of estrone abolished its binding to the estrogen receptors. RGC-5 cells (an established clonal rat retinal ganglion cell line) and rat retinas were shown to predominantly express estrogen receptor alpha, with minimal detectable levels of estrogen receptor beta. The affinity of the synthetic compounds for estrogen receptors was as follows: ZYC-3 < ZYC-10 < ZYC-1. An in vitro model of glutamate-induced RGC-5 cell death was used. Glutamate treatment resulted in 50-60% RGC-5 cell death with respect to control untreated cells. 17beta-estradiol and the three estrogen analogues (0.5 to 1.0 microM) protected the RGC-5 cells against glutamate cytotoxicity. The efficacy of neuroprotection by the estrogen analogues was as follows: ZYC-3 > ZYC-1 > ZYC-10. EC(50) values for inhibition of TBARS levels were as follows: ZYC-3 > ZYC-10 > ZYC-1. Furthermore, these compounds worked independent of estrogen receptors, as inclusion of 100 nM ICI 182,780 did not reverse their neuroprotective properties against glutamate insult. These compounds seem to affect neuroprotection via pathways independent of the classical estrogen receptors. The data support the hypothesis that estrogen analogues may be useful in the treatment of neurodegenerative diseases, particularly in neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.     

Laccase treatment impairs bisphenol A-induced cancer cell proliferation affecting estrogen receptor alpha-dependent rapid signals

A wide variety of environmental contaminants exert estrogenic actions in wildlife, laboratory animals, and in human beings through binding to nuclear estrogen receptors (ERs). Here, the mechanism(s) of bisphenol A (BPA) to induce cell proliferation and the occurrence of its bioremediation by treatment with laccase are reported. BPA, highly present in natural world and considered as a model of environmental estrogen action complexity, promotes human cancer cell proliferation via ERalpha-dependent signal transduction pathways. Similar to 17beta-estradiol, BPA increases the phosphorylation of both extracellular regulated kinase and AKT. Specific inhibitors of these kinase completely block the BPA effect on cancer cell proliferation. Notably, high BPA concentrations (i.e., 0.1 and 1 mM) are cytotoxic even in ERalpha-devoid cancer cells, indicating that an ERalpha-independent mechanism participates to BPA-induced cytotoxicity. On the other hand, BPA oxidation by laccase impairs the binding of this environmental estrogen to ERalpha loosing at all ERalpha-dependent effect on cancer cell proliferation. Moreover, the laccase-catalyzed oxidation of BPA reduces the BPA cytotoxic effect. Thus, laccase appears to impair BPA action(s), representing an invaluable bioremediation enzyme.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

Daphnetin, a Natural Coumarin Derivative, Provides the Neuroprotection Against Glutamate-Induced Toxicity in HT22 Cells and Ischemic Brain Injury

Daphnetin (DAP), a coumarin derivative, has been reported to have multiple pharmacological actions including analgesia, antimalarial, anti-arthritic, and anti-pyretic properties. It is unclear whether DAP has neuroprotective effects on ischemic brain injury. In this study, we found that DAP treatment (i.c.v.) reduced the infarct volume at 24 h after ischemia/reperfusion injury and improved neurological behaviors in a middle cerebral artery occlusion mouse model. Moreover, we provided evidences that DAP had protective effects on infarct volume in neonate rats even it was administrated at 4 h after cerebral hypoxia/ischemia injury. To explore its neuroprotective mechanisms of DAP, we examined the protection of DAP on glutamate toxicity-induced cell death in hippocampal HT-22 cells. Our results demonstrated that DAP protected against glutamate toxicity in HT-22 cells in a concentration-dependent manner. Further, we found that DAP maintained the cellular levels of glutathione and superoxide dismutase activity, suggesting the anti-oxidatant activity of DAP. Since DAP has been used for the treatment of coagulation disorder and rheumatoid arthritis for long time with a safety profile, DAP will be a promising agent for the treatment of stroke.     

Antioxidant activity and neuroprotective effects of zolpidem and several synthesis intermediates

Structural relationship between the antioxidant melatonin and the non-benzodiazepine hypnotic zolpidem (ZPD) suggests possible direct antioxidant and neuroprotective properties of this compound. In the present work, these effects were analyzed for zolpidem and four of its synthesis intermediates. In vitro assays include lipid peroxidation and protein oxidation studies in liver and brain homogenates. Intracellular antioxidant effects were analyzed by evaluation of free radical formation prevention in HT-22 hippocampal cells treated with glutamate 10mM and measured by flow cytometer DCF fluorescence. The neuroprotective effect of these compounds was evaluated as neuronal death prevention of HT-22 cells treated with the same concentration of glutamate. Zolpidem was found to prevent induced lipid peroxidation in rat liver and brain homogenates showing figures similar to melatonin, although it failed to prevent protein oxidation. ZPD-I was the most effective out of the several zolpidem intermediates studied as it prevented lipid peroxidation with an efficiency higher than melatonin or zolpidem and with an effectiveness similar to estradiol and trolox. ZPD-I prevents protein oxidation, which trolox is known to be unable to prevent. When cellular experiments were undertaken, ZPD-I prevented totally the increase of intracellular free radicals induced by glutamate 10mM in culture medium for 12h, while zolpidem and ZPD-III partially prevented this increase. Also the three compounds protected hippocampal neurons from glutamate-induced death in the same conditions, being their comparative efficacy, ZPD-III > ZPD-I = ZPD.     

Bisphenol a inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.     

Conjugation of catechins with cysteine generates antioxidant compounds with enhanced neuroprotective activity

Antioxidant compounds derived from the conjugation of (-)-epicatechin and (-)-epicatechin 3-O-gallate with cysteine and cysteine derivatives protected HT-22 nerve cells (EC50 between 36 and 65 microM) from death triggered by glutamate while underivatized (-)-epicatechin was almost inactive (EC50=610 microM). Differences in free radical scavenging capacity (DPPH assay) could not account for the improvement in neuroprotective activity upon derivatization of (-)-epicatechin with thiols. Moreover, while the gallate-containing compounds are more efficient radical scavengers than their non-galloylated counterparts, they are only equally or less potent as neuroprotective agents. Although all of the conjugates were able to scavenge mitochondrially generated reactive oxygen species (ROS) inside the cells, the majority of their neuroprotective activity appeared to be dependent upon their ability to maintain glutathione levels. These results suggest that a mechanism other than ROS scavenging is involved in the neuroprotective action exerted by the epicatechin conjugates.     

Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells

Neuronal cell death caused by oxidative stress is common in a variety of neural diseases and can be investigated in detail in cultured HT22 neuronal cells, where the amino acid glutamate at high concentrations causes glutathione depletion by inhibition of the glutamate/cystine antiporter system, intracellular accumulation of reactive oxygen species (ROS) and eventually oxidative stress-induced neuronal cell death. Using this paradigm, we have previously reported that resveratrol (3,5,4′-trans-trihydroxystilbene) protects HT22 neuronal cells from glutamate-induced oxidative stress by inducing heme oxygenase (HO)-1 expression. Piceatannol (3,5,4′,3′-trans-trihydroxystilbene), which is a hydroxylated resveratrol analog and one of the resveratrol metabolites, is estimated to exert neuroprotective effect similar to that of resveratrol. The aim of this study, thus, is to determine whether piceatannol, similarly to resveratrol, would protect HT22 neuronal cells from glutamate-induced oxidative stress. Glutamate at high concentrations induced neuronal cell death and ROS formation. Piceatannol reduced glutamate-induced cell death and ROS formation. The observed cytoprotective effect was much higher when HT22 neuronal cells were pretreated with piceatannol for 6 or 12 h prior to glutamate treatment than when pretreated for 0.5 h. Piceatannol also increased HO-1 expression and HO activity via its activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, neuroprotective effect of piceatannol was partly (but not completely) abolished by either down-regulation of HO-1 expression or blockage of HO-1 activity. Taken together, our results suggest that piceatannol, similar to resveratrol, is capable of protecting HT22 neuronal cells against glutamate-induced cell death, at least in part, by inducing Nrf2-dependent HO-1 expression.     

Neuroprotective effects of naturally occurring biflavonoids

We examined neuroprotective effects of naturally occurring biflavonoids on oxidative stress-induced and amyloid beta peptide-induced cell death in neuronal cells. Among the nine biflavonoids tested, amentoflavone, ginkgetin, and isoginkgetin exhibited strong neuroprotection against cytotoxic insults induced by oxidative stress and amyloid beta, suggesting their therapeutic potential against neurodegenerative diseases, including ischemic stroke and Alzheimer's disease.     

AIF depletion provides neuroprotection through a preconditioning effect

Previous studies established a major role for apoptosis inducing factor (AIF) in neuronal cell death after acute brain injury. For example, AIF translocation from mitochondria to the nucleus determined delayed neuronal death, whereas reduced AIF expression provided neuroprotective effects in models of cerebral ischemia or brain trauma. The question remains, however, why reduced AIF levels are sufficient to mediate neuroprotection, since only very little AIF translocation to the nucleus is required for induction of cell death. Thus, the present study addresses the question, whether AIF gene silencing affects intrinsic death pathways upstream of nuclear translocation at the level of the mitochondria. Using MTT assays and real-time cell impedance measurements we confirmed the protective effect of AIF siRNA against glutamate toxicity in immortalized mouse hippocampal HT-22 neurons. Further, AIF siRNA prevented glutamate-induced mitochondrial fragmentation and loss of mitochondrial membrane potential. The protection of mitochondrial integrity was associated with preserved ATP levels, attenuated increases in lipid peroxidation and reduced complex I expression levels. Notably, low concentrations of the complex I inhibitor rotenone (20?nM), provided similar protective effects against glutamate toxicity at the mitochondrial level. These results expose a preconditioning effect as a mechanism for neuroprotection mediated by AIF depletion. In particular, they point out an association between mitochondrial complex I and AIF, which regulate each other's stability in mitochondria. Overall, these findings postulate that AIF depletion mediates a preconditioning effect protecting neuronal cells from subsequent glutamate toxicity through reduced levels of complex I protein.     

Propofol hemisuccinate protects neuronal cells from oxidative injury

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 alpha-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.     

Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway. Implications for neuroprotection

We hypothesize that in neurodegenerative disorders such as Alzheimer's 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-3beta (GSK3beta), a pathway important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a time-dependent inactivation of GSK3beta by phosphorylation at serine 9. Blocking FGF1 receptors with heparinase reduced this effect. The effects of FGF1 on GSK3beta 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 beta-catenin translocation into the nucleus. On the other hand, infection with wild-type GSK3beta recombinant adenovirus-associated virus increased activity of GSK3beta and cell death, both of which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity was dependent on GSK3beta inactivation by the PI3K-Akt but was independent of ERK. Taken together these results suggest that neuroprotective effects of FGF1 might involve inactivation of GSK3beta by a pathway involving activation of the PI3K-Akt cascades.     

Neuroprotective actions of deferiprone in cultured cortical neurones and SHSY-5Y cells

Alzheimer's disease (AD) is a common neurodegenerative disorder, but the initiating molecular processes contributing to neuronal death are not well understood. AD is associated with elevated soluble and aggregated forms of amyloid beta (Abeta) and with oxidative stress. Furthermore, there is increasing evidence for a detrimental role of iron in the pathogenic process. In this context, iron chelation by compounds such as 3-hydroxypyridin-4-one, deferiprone (Ferriprox) may have potential neuroprotective effects. We have evaluated the possible neuroprotective actions of deferiprone against a range of AD-relevant insults including ferric iron, H(2)O(2) and Abeta in primary mouse cortical neurones. We have investigated the possible neuroprotective actions of deferiprone (1, 3, 10, 30 or 100 microM) in primary neuronal cultures following exposure to ferric iron [ferric nitrilotriacetate (FeNTA); 3 and 10 microM], H(2)O(2) (100 microM) or Abeta1-40 (3, 10 and 20 microM). Cultures were treated with deferiprone or vehicle either immediately or up to 6 h after the insult in a 24-well plate format. In order to elucidate a possible neuroprotective action of deferiprone against Parkinson's disease relevant insults another group of experiments were performed in the human neuroblastoma catecholaminergic SHSY-5Y cell line. SHSY-5Y cells were treated with MPP(+) iodide, the active metabolite of the dopaminergic neurotoxin MPTP and the neuroprotective actions of deferiprone evaluated. Cytotoxicity was assessed at 24 h by lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide turnover (FeNTA and hydrogen peroxide) and morphometric analysis of cell viability by Hoechst 33324/propidium iodide (FeNTA, Abeta and MPP(+)) or 6-carboxyfluorescein diacetate and annexin V-Cy3 (Abeta). The present study demonstrates that deferiprone protects against FeNTA, hydrogen peroxide, MPP(+) and Abeta1-40-induced neuronal cell death in vitro, which is consistent with previous in vitro and in vivo studies that have demonstrated similar protection with other iron chelators.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Regulation of estrogen receptors in primary cultured hepatocytes of the amphibian Xenopus laevis as estrogenic biomarker and its application in environmental monitoring

The present study aims to introduce the regulation of estrogen receptors (ER) in primary cultured hepatocytes of the amphibian Xenopus laevis as a further potential estrogenic biomarker. Time courses of free ER in cell cultures treated with 17beta-estradiol (E2), nonylphenol (NP), and bisphenol A (BPA) were determined by means of radioreceptorassay (RARA). All compounds led to an immediate drop of free ER followed by a significant increase. The estrogen specific induction of ER-mRNA in vitro during time course was verified by using semiquantitative RT-PCR demonstrating greatest differences after 36 h. Dose-response curves of ER-mRNA for E2, NP, and BPA revealed that E2 possessed highest estrogenicity starting at 10(-9) M, while NP and BPA induced significant increases at 10(-8) and 10(-7) M, respectively. Extracts of the river Alb were subjected to RARA for ER binding to cytosolic liver fraction as well as to primary cultured hepatocytes for assessment of ER-mRNA induction. The results by RARA demonstrated clearly that binding to ER was highest in sewage treatment plant effluents and increased during the course of the river. These findings could be correlated with induction of ER-mRNA levels in vitro indicating that both techniques are suitable for application in monitoring of estrogenic EDC.