Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway.

Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.

Brain aging phenomena in migrating sockeye salmon Oncorhynchus nerka nerka.

Aging, a process occurring in all vertebrates, is closely related to a loss in physical and functional abilities. There is widespread interest in clarifying the relevance of environmental, metabolic, and genetic factors for vertebrate aging. In the Pacific salmon a dramatic example of aging is known. Looking for changes in the salmon brain, perhaps even in the role of initiating the aging processes, we investigated several biochemical parameters that should reflect brain functional activity and stress response such as the neurotransmitters dopamine, and serotonin, and two of their respective metabolites 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole acetic acid, as well as glutathione, glutathione disulfide, and the extent of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling. The aging of migrating sockeye salmon (Oncorhynchus nerka nerka) is accompanied by gradual increase in dopamine and serotonin turnover and a gradual decrease of brain total protein and glutathione levels. There appears to be an increased need for detoxification of reactive biological intermediates since activities of superoxide dismutase and catalase increase with age. However, our data do not support a major increase in apoptotic cell death during late aging but rather implicate an age related downward regulation of protein and glutathione synthesis and proteolysis increasing the need for autophagocytosis or heterophagocytosis in the course of cell death.

(r)-, but not (s)-alpha lipoic acid stimulates deficient brain pyruvate dehydrogenase complex in vascular dementia, but not in Alzheimer dementia.

In dementia of Alzheimer type (DAT), cerebral glucose metabolism is reduced in vivo, and enzymes involved in glucose breakdown are impaired in post-mortem brain tissue. Pyruvate dehydrogenase complex activity (PDHc) is one of the enzymes known to be reduced, while succinate dehydrogenase activity (SDH), another enzyme of oxidative glucose metabolism is unchanged. In dementia of vascular type (DVT), variable changes in glucose metabolism have been demonstrated in vivo, while changes of enzyme activities in post-mortem brain tissue are unknown. Here, PDHc and SDH activity were stimulated with each of the two stereoisomers of alpha lipoic acid in post-mortem parietal brain cortex of patients with DAT, DVT, and one case of Pick's disease and compared to stimulation effects in a control group, matched for age, sex, post-mortem delay, and storage time of brain tissue. PDHc in DAT and DVT, but not in Pick's disease was reduced. PDHc activity could be slightly stimulated by 10 micro M of the physiological stereoisomer (r)-alpha-lipoic acid, in controls and DVT (possibly also in Pick's disease), but not in DAT. In all groups investigated SDH was activated by 100 micro M and 1 mM of both isomers of alpha-lipoic acid, whereas 10 mM of both stereoisomers of alpha-lipoic acid caused an inhibition of both, PDHc and SDH activity. The loss of basal and of (r)-alpha-lipoic acid stimulated PDHc activity indicate that a functional or structural impairment of PDHc may exist in DAT and DVT which is not merely attributable to loss of mitochondria since basal and stimulated SDH activities are similar in controls, DVT and DAT, thus indicating selective vulnerability of PDHc.

Methylmercury induces neurite degeneration in primary culture of mouse dopaminergic mesencephalic cells.

Methylmercury cation (MeHg) is an hazardous environmental pollutant with neurotoxic action. Little is known about the effects of MeHg on catecholaminergic neurons. In the present study we have used epifluorescence microscopy and confocal microscopy to investigate the alterations induced by MeHg in primary DA (dopaminergic) cells isolated from the ventral mesencephalon of CD-1 embryonic mice and cultured for six days in vitro. DA cells were identified in the multi-culture by immunocytochemistry using a tyrosine-hydroxylase antibody. The morphometric analysis of DA neurons exposed to 1 microM MeHg demonstrated a striking decrease in the number of neurites, indicative of cytoskeletal alteration. In addition, DA neurons displayed cell shrinkage and a significant increase of nuclei with chromatin condensation. Based on these results it is concluded that MeHg is highly toxic to primary DA neurons.

Unaltered brain levels of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal in Alzheimer's disease.

In recent years, an important role for the pathogenesis of Alzheimer's disease (AD) has been ascribed to oxidative stress. Trans-4-hydroxy-2-nonenal, a product of lipid peroxidation, forms stable adducts with a variety of nucleophilic substituents such as thiols or amino moieties. Here, we report the quantification of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp) using the specific and very sensitive method of 32P-postlabeling of deoxyguanosine adducts derived from nuclear DNA in neuron rich areas of the hippocampus, the parietal cortex, and the cerebellum of postmortem brains from patients with AD and age matched controls. Adduct levels were highest in the hippocampus, followed by the cerebellum and parietal cortex irrespective of the disease. Neither age, postmortem delay time, gender, nor the extent of neurofibrillary deposits affected tissue adduct levels in the brain areas examined. Although distinctively present in the human brain, the level of HNE-dGp adducts appears not to be useful as a biomarker for AD.

A highly sensitive method for the determination of protein bound 3,4-dihydroxyphenylalanine as a marker for post-translational protein hydroxylation in human tissues ex vivo.

A highly sensitive, specific and tissue-independent method is described to evaluate oxidative stress-mediated protein hydroxylation in red blood cells, frontal cortex, and liver by HPLC separation and electrochemical detection of protein-bound 3,4-dihydroxyphenylalanine (DOPA) following gas-phase amino acid hydrolysis of tissue protein extracts containing exclusively proteins larger than 3 kDa. Simultaneous measurement of protein tyrosine (Tyr) content using fluorescence detection results in a tissue specific DOPA/Tyr ratio that may reflect oxidative stress-mediated protein modifications in disease, or following the exposure to oxidative stress-inducing agents.

Monoamine oxidase-inhibition and MPTP-induced neurotoxicity in the non-human primate: comparison of rasagiline (TVP 1012) with selegiline.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in man and non-human primates. Monoamine-oxidase B (MAO-B) has been reported to be implicated in both MPTP-induced parkinsonism and Parkinson's disease, since selegiline (L-deprenyl), an irreversible MAO-B inhibitor, prevents MPTP-induced neurotoxicity in numerous species including mice, goldfish and drosophyla. However, one disadvantage of this substance relates to its metabolism to (-)-methamphetamine and (-)-amphetamine. Rasagiline (R-(+)-N-propyl-1-aminoindane) is a novel irrevesible MAO-B-inhibitor, which is not metabolized to metamphetamine and/or amphetamine. The present study compared the effects of high doses of selegiline and rasagiline (10 mg/kg body weight s.c.) on MPTP-induced dopaminergic neurotoxicity in a non-human primate (Callithrix jacchus) model of PD. Groups of four monkeys were assigned to the following six experimental groups: Group I: Saline, Group II: Selegiline/Saline, Group III: Rasagiline/Saline, Group IV: MPTP/Saline, Group V: Rasagiline/MPTP, Group VI: Selegiline/MPTP. Daily treatment with MAO-B-inhibitors (either rasagiline or selegiline, 10 mg/kg body weight s.c.) was initiated four days prior to MPTP-exposure (MPTP-HCl, 2 mg/kg body weight subcutaneously, separated by an interval of 24 hours for a total of four days) and was continued until the end of the experiment, i.e. 7 days after the cessation of the MPTP-injections, when animals were sacrificed. MPTP-treatment caused distinct behavioural, histological, and biochemical alterations: 1. significant reduction of motor activity assessed by clinical rating and by computerized locomotor activity measurements; 2. substantial loss (approx. 40%) of dopaminergic (tyrosine-hydroxylase-positive) cells in the substantia nigra, pars compacta; and 3. putaminal dopamine depletion of 98% and its metabolites DOPAC (88%) and HVA (96%). Treatment with either rasagiline or selegiline markedly attenuated the neurotoxic effects of MPTP at the behavioural, histological, and at the biochemical levels. There were no significant differences between rasagiline/MPTP and selegiline/MPTP-treated animals in respect to signs of motor impairment, the number of dopaminergic cells in the substantia nigra, and striatal dopamine levels. As expected, both inhibitors decreased the metabolism of dopamine, leading to reduced levels of HVA and DOPAC (by >95% and 45% respectively). In conclusion, rasagiline and selegiline at the dosages employed equally protect against MPTP-toxicity in the common marmoset, suggesting that selegiline-derived metabolites are not important for the neuroprotective effects of high dose selegiline in the non-human MPTP-primate model in the experimental design employed. However, unexpectedly, high dose treatment with both MAO-inhibitors caused a decrease of the cell sizes of nigral tyrosine hydroxylase positive neurons. It remains to be determined, if this histological observation represents potential adverse effects of high dose treatment with monoamine oxidase inhibitors.

cAmp signaling cascade: a promising role in ethanol tolerance and dependence.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Subhash C. Pandey and Toshikazu Saito. The presentations were (1) Action of ethanol on cAMP signaling pathways, by M. Yoshimura; (2) Alterations in the G protein adenylyl cyclase system and their mRNA levels in alcoholics, by H. Sohma; (3) The role of the CREB gene transcription factor in ethanol dependence and preference, by Subhash C. Pandey; and (4) The efficacy of adenylyl cyclase signal transduction to the nucleus in primary alcoholics, by M. E. Götz.

Chronic alcohol consumption and cerebral indices of oxidative stress: is there a link?

BACKGROUND: It is still difficult to define the biochemical mechanisms that cause alterations in neuronal function and plasticity and neuronal cell loss in the brains of alcohol-dependent patients. METHODS: To evaluate the extent of cerebral alcohol-induced oxidative stress ex vivo, we investigated the levels of glutathione (GSH), its oxidation product glutathione disulfide (GSSG, produced by GSH-peroxidases), and the activities of catalase and superoxide dismutases (SOD). In addition, selected brain regions from up to 22 subjects (versus controls) were studied post mortem to compare the amount of oxidized DNA-base 8-hydroxy-2'-deoxyguanosine (8-OHdG) with levels of deoxyguanosine (dG) in mitochondrial and nuclear DNA. RESULTS: The most prominent findings showed significantly decreased GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in the corpus mamillare and cerebellum, which appeared due to an increase in GSSG caused by chronic alcohol intake. Catalase activity was increased in only the frontal cortex, whereas decreased catalase activity was found in the corpus callosum. In contrast, neither copper-zinc-superoxide dismutase (CuZnSOD) and manganese-superoxide dismutase (MnSOD) activities nor 8-OHdG/dG molar ratios were altered, although a tendency toward higher OHdG/dG ratios in temporal and parietal cortex from alcohol-dependent patients could be detected when mitochondrial DNA was analyzed selectively. CONCLUSIONS: We propose that decreased brain GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in alcohol-dependent patients may reflect neural impairment due to increased peroxide production after chronic alcohol consumption. However, future experiments, investigating the activities of enzymes and cofactors involved in GSH synthesis and metabolism in the human brain, will have to validate the specificity of these results for oxidative stress.

Increased levels of calcium-sensitive adenylyl cyclase subtypes in the limbic system of alcoholics: evidence for a specific role of cAMP signaling in the human addictive brain.

We examined the amounts of several adenylyl cyclase (AC) isoforms and of cAMP-response element binding protein (CREB) in alcoholic and control brains. Immunoreactivity of type I AC was significantly increased in alcoholic nucleus accumbens and corpus amygdaloideum. Immunoreactivity of type VIII AC was also increased in alcoholic corpus amygdaloideum and hippocampus. CREB immunoreactivities were unchanged. These findings indicate that the brain-region specific increase of Ca(2+)-sensitive AC may contribute to the pathophysiology of alcoholism.

Selegiline completely restores choline acetyltransferase activity deficits in simian immunodeficiency infection.

Human immunodeficiency virus (HIV) infection is associated with a progressive dementia, in addition to motor and behavioural deficits. Cognitive deterioration and motor impairments have been observed also in simian immunodeficiency virus (SIV)-infected monkeys, an animal model for HIV infection. We found recently that choline acetyltransferase activity is markedly reduced in brains of SIV-infected monkeys. We report now that selegiline, completely restores the reduced choline acetyltransferase activity which encourages for a meaningful anti-dementia therapeutic strategy.

Immunoreactivity of cAMP response element binding protein is not altered in the post-mortem cerebral cortex or cerebellum of alcoholics.

We examined amounts of cAMP response element binding protein (CREB) and its phosphorylated form in post-mortem frontal and temporal cortices and cerebella from alcoholics and controls by immunoblotting. No significant differences were observed in the levels of these proteins in each brain region, suggesting that the assumed neuroadaptations to chronic ethanol intake may not be reflected by quantitative alterations of CREB in alcoholic brain.

Hippocampal level of neural specific adenylyl cyclase type I is decreased in Alzheimer's disease.

Previous studies reported disruption of adenylyl cyclase (AC)-cyclic AMP (cAMP) signal transduction in brain of Alzheimer's disease (AD). We also demonstrated that basal and stimulated AC activities in the presence of calcium and calmodulin (Ca(2+)/CaM) were significantly decreased in AD parietal cortex. In the present study, we examined the amounts of Ca(2+)/CaM-sensitive types I and VIII AC, and Ca(2+)/CaM-insensitive type VII AC in the postmortem hippocampi from AD patients and age-matched controls using immunoblotting. The specificities of the anti-type VII and VIII AC antibodies were confirmed by preabsorption with their specific blocking peptides. We observed a significant decrease in the level of type I AC and a tendency to decrease in the level of type VIII AC in AD hippocampus. On the other hand, the level of type VII AC showed no alteration between AD and controls. A body of evidence from the studies with invertebrates and vertebrates suggests that types I and VIII AC may play an essential role in learning and memory. Our finding thus firstly demonstrated that a specific disruption of the Ca(2+)/CaM-sensitive AC isoforms is likely involved in the pathophysiology in AD hippocampus.

Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death.

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication.

Altered redox state of platelet coenzyme Q10 in Parkinson's disease.

BACKGROUND: The reduced form of coenzyme Q10 (CoQ10) acts as a lipophilic antioxidant and participates in electron and proton transport of the respiratory chain in the inner mitochondrial membrane. An alteration in CoQ10 redox state may thus reflect a change in membrane electron transport and the effectiveness of defense against toxic reactive oxygen species such as hydrogen peroxide and superoxide. In Parkinson's disease alterations in the activities of complex I have been reported in substantia nigra and platelets. Deficiency of mitochondrial enzyme activities could affect electron transport which might be reflected by the platelet CoQ10 redox state. METHOD: We have determined concentrations of the reduced and oxidized forms of CoQ10 and the activity of monoamine oxidase B in platelets isolated from parkinsonian patients and age- and gender-matched controls. RESULTS: Platelet CoQ10 redox ratios (reduced CoQ10 to oxidized CoQ10) and the ratio of the reduced form, compared with total platelet CoQ10, were significantly decreased in de novo parkinsonian patients. Platelet CoQ10 redox ratios were further decreased by L-DOPA treatment (not significant), whilst selegiline treatment partially restored CoQ10 redox ratios. Monoamine oxidase activities in non-selegiline treated patients were similar to controls. INTERPRETATION: Our results either suggest an impairment of electron transport or a higher need for reduced forms of CoQ10 in the platelets of even de novo parkinsonian patients. However, the CoQ10 redox ratio was not correlated to disease severity, as determined by the Hoehn and Yahr PD disability classification, suggesting that this parameter may not be useful as a peripheral trait marker for the severity of PD but as an early state marker of PD.

Radical scavenging compound J 811 inhibits hydrogen peroxide-induced death of cerebellar granule cells.

Oxidative stress is considered to be an important pathophysiological condition to promote cell death in a broad variety of disorders, such as cardiovascular and neurodegenerative diseases. Scavestrogens, structurally derived from estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the so-called scavestrogen estra-1,3,5(10),8-tetraene-3,17alpha-diol, J 811, was tested using rat cerebellar granule cells (CGCs) exposed to 25 or 50 microM hydrogen peroxide (H2O2). H2O2-induced apoptotic cell death was detected by the appearance of high molecular weight DNA fragments and nuclear condensation. The addition of J 811 before or shortly after the exposure to H2O2 prevented CGC apoptosis in a dose-dependent manner. The estrogen receptor antagonist ICI 182.780 failed to prevent the protective effect of J 811, suggesting that the latter is not dependent on estrogen receptor activation. The lack of protection against apoptosis caused by colchicine suggests that J 811 is neither interfering with the activation of caspase-3, nor acting downstream of caspase-3. Therefore, the protective effect observed against H2O2 seems to be upstream caspases activation, pointing to a scavenging action of J 811. Thus the scavestrogen J 811 is a powerful antioxidant able to interfere with radical-mediated cell death and is potentially useful in diseases where reactive oxygen species are involved.

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type.

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.

Scavestrogens protect IMR 32 cells from oxidative stress-induced cell death.

Oxidative stress is considered an important pathophysiological mechanism contributing to promote cell death in a broad variety of diseases including cardiovascular and neurodegenerative disorders. The so-called scavestrogens J811 and J861, structurally derived from 17alpha-estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the scavestrogens J811 and J861 against Fenton reagent-induced cell damage (50 microM FeSO4 plus 200 microM H2O2) were compared with those of 17alpha- and 17beta-estradiol. Cell viability studies using Trypan blue staining showed that estradiols and scavestrogens at concentrations ranging from 0.1 to 10 microM are able to protect IMR 32 neuroblastoma cells from Fenton-mediated death. In addition, these compounds decreased lipid peroxidation measured as thiobarbituric acid reactive substances and renormalize oxidative stress-increased intracellular glutathione levels. When given 6 h after the toxic stimulus, J811 and J861 rescued 60% of cells, whereas 17alpha- and 17beta-estradiol were ineffective. These results suggest that the scavestrogens J811 and J861 are powerful antioxidants capable of interfering with radical-mediated cell death in diseases known to be aggravated by reactive oxygen species. Such compounds may be useful in the development of novel treatments for stroke or neurodegenerative disorders.

Chronic TVP-1012 (rasagiline) dose--activity response of monoamine oxidases A and B in the brain of the common marmoset.

The stereospecific form of the known acetylenic mechanism-based MAO-inhibitor AGN1135 (Rasagiline, TVP-1012) is devoid of sympathomimetic amphetamine-like properties. To evaluate the efficiency and selectivity of subcutaneous injections of TVP-1012 (dose range from 0.01 up to 10 mg/kg for 7 days) the activities of monoamine oxidases A and B (MAO-A,-B) were determined in different brain regions of the common marmoset. At a dose of 0.1 mg/kg TVP-1012, almost 80% of MAO-B activity is inhibited in all brain regions investigated (prefrontal and occipital cortex, cerebellum, caudate nucleus, putamen, nucleus accumbens). In contrast, MAO-A is not inhibited in putamen and nucleus accumbens. However, by increasing the TVP-1012 dose to 0.5 mg/kg, MAO-A is inhibited to a significant extent as well, concomitant to total inhibition of MAO-B. The results obtained indicate that TVP-1012 irreversibly inhibits both types of MAO in the common marmoset with selectivity for MAO-B at doses less than 0.5 mg/kg. TVP-1012 could be useful in studies requiring selective MAO-B inhibition without concomitant sympathomimetic amphetamine-like effects and could thus be of therapeutic interest for Parkinson's disease and retarded depression.