Restorative Neurology and Neuroscience - Volume 12, issue 2-3

Authors: Manev, Hari | Costa, Erminio | Flaherty, Joseph A.

Article Type: Other

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 57-58, 1998

Authors: Richter, Christoph

Article Type: Research Article

Abstract: This article emphasizes the importance of mitochondria, the cellular ATP level, and the liberation of certain mitochondrial proteins for the execution phase of apoptosis. Destabilization of mitochondria results in release of these proteins. Oxidative stress and altered cellular Ca2+ homeostatis, considered to be mediators of apoptosis, synergistically decrease the mitochondrial membrane potential and lower the cellular ATP level. Conversely, stabilization of the mitochondrial membrane potential, e.g., by the protooncogene bcl-2, prevents cell …death. An important process underlying mitochondrial destabilization is oxidant-induced mitochondrial Ca2+ release followed by re-uptake ("Ca2+ cycling"). Tumor necrosis factor-a induces oxygen radicals in mitochondria through ceramides, and the recently discovered mitochondrial nitric oxide synthase profoundly stimulates Ca2+ release from mitochondria through formation of nitrogen monoxide and peroxynitrite. Show more

Keywords: Ca2+, ceramides, cytochrome c, membrane potential, reactive nitrogen, reactive oxyogen

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 59-62, 1998

Authors: Kong, Ah-Ng Tony | Yu, Rong | Lei, Wei | Mandlekar, Sandhya | Tan, Tse-Hua | Ucker, David S.

Article Type: Research Article

Abstract: Chemical-induced oxidative stress to a cell can signal many cellular responses which include proliferation, differentiation, hemeostasis, apoptosis or necrosis. To better understand the underlying molecular mechanisms after exposure to chemicals, we investigated the signal transduction pathways, in particular the mitogen-activated protein kinase (MAPK) pathway and the ICE/Ced-3 protease (caspase) pathway, activated by different agents. Butylated hydroxyanisol (BHA) and its metabolite, t-butyl-hydroquinone (tBHQ), both are well known phenolic antioxidants used in food preservatives, …strongly activated c-Jun N-terminal kinase 1 (JNK1) and/or extracellular signal-regulated protein kinase 2 (ERK2) in a dose- and time-dependent fashion. Pretreatment with free radical scavengers N-acetyl-L-cysteine (NAC), glutathione (GSH), or vitamin E, inhibited ERK2 activation and, to a much lesser extent, JNK 1 activation by BHA and tBHQ, implicating the role of oxidative stress. Under conditions where JNK1 and ERK2 were activated, BHA also activated transcription factors nuclear factor kappa B (NF-?B), activated-protein-1 (AP-1), and anti-oxidant response element (ARE), leading to induction of genes such as c-jun, and c-fos. At relatively high concentrations, BHA and tBHQ stimulated proteolytic activity of ICE/Ced3 cysteine proteases, and caused apoptosis, which was blocked by pretreatment with NAC. Further increase in concentrations lead to rapid cell death predominantly occurred via necrosis. Some naturally occurring phytochemicals, such as phenylethyl isothiocyanate (PEITC), green tea polyphenols (GTP), and sulfarophane, which have been shown to be potent inducers of Phase II enzymes, also differentially regulated the activities of JNK, ERK, or CPP-32, in a time- and dose-dependent manner. Our data, together with the work of others, enable us to propose a model in which low concentrations of these chemicals (e.g., BHA, PEITC) activate MAPKs leading to induction of gene expression (e.g., c-jun, c-fos, GSI) which may protect the cells against toxic insults and enhance cell survival. At relatively high concentrations, these agents activated both MAPKS, and the ICE/Ced-3 caspase pathway, leading to apoptosis. The exact mechanisms by which MAPK and caspases are activated by these agents are currently unknown, but may involve oxidative modification of glutathione (GSH) and/or protein thiols, and/or generation of secondary messengers, ceramide and calcium, which further activate downstream events. Taken together, our results suggest that chemicals including phenolic antioxidants activate MAPK pathways which may lead to the induction of genes producing protection and survival mechanisms, as well as the ICE/Ced-3 protease pathway, leading to apoptosis. The balancing amongst these pathways may dictate the fate of the cells upon exposure to chemicals. Show more

Keywords: oxidative stess, apoptosis, caspase, MAPK, ERK, JNK, BHA

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 63-70, 1998

Authors: Kiedrowski, Lech

Article Type: Research Article

Abstract: The hypothesis that a destabilization of mitochondrial function during neuronal exposure to excitatory amino acids may be involved in the mechanism of neuronal death was examined. The mitochondrial membrane potential (??m) and the cytoplasmic Ca2+ concentration ([Ca2+]c) were monitored simultaneously in single cultured rat cerebellar granule cells (CGCs) loaded with tetramethylrhodamine methyl ester (TMR) and fura-2; CGCs were depolarized with K+, or exposed to excitotoxic doses of glutamate or kainate, and viability of the same neurons was studied for 24-30 h. This approach made it possible to single out the neurons that died, and to describe the changes in ??m …and [Ca2+]c that were characteristic for these neurons. Exposure to glutamate caused an increase in [Ca2+]c that was associated with a decrease in the mitochondrial TMR fluorescence, which indicates a decrease in ??m. The neurons that failed to restore ??m following glutamate withdrawal, also failed to restore low [Ca2+]c, and later died. Although a similar number of neurons died following kainate exposure as did after glutamate exposure, the kainate-elicited neuronal death resulted not from the collapse of ??m but from an excessive neuronal swelling, which led to rupture of the plasma membrane. Depolarzation with K+ was not neurotoxic and caused only a minor decrease in TMR fluorescence. These results indicate that in vitro glutamate and kainate destroy neurons by different mechanisms: glutamate by a failure to restore ??m following the exposure, and kainate by an osmotic lesion of the plasma membrane. Show more

Keywords: glutamte, kainate, NMDA, mitochondria, membrane potential, depolarization, calcium, sodium, neurons, excitotxicity, apoptosis, neuronal death

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 71-79, 1998

Authors: Manev, H. | Uz, T. | Qu, T.

Article Type: Research Article

Abstract: 5-Lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) is the enzyme responsible for the first step in the formation of inflammatory leukotrienes from arachidonic acid. 5-LO is expressed in hippocampal neurons. Increased formation of leukotrienes was found in the hippocampus of rats in which seizures were induced by a glutamate receptor agonist, kainate. Expression of the 5-LO gene can be stimulated by vitamin D3 and suppressed by the pineal hormone melatonin. Here we hypothesize that …kainate also stimulates 5-LO expression in the hippocampus. Kainate was injected intraperitoneally (10 mg/kg). Rats were sacrificed 3 hr later and their hippocampi were dissected and total RNA was extracted and processed for quantitative reverse transcription/polymerase chain reaction (RT-PCR) assay of 5-LO and cyclophilin (cyc) mRNAs. Mutated primers were used as internal standards to assay attomol quantities of these two specific mRNAs per microgram of total RNA. Fixed hippocampal slices were processed for 5-LO immunostaining and Nissl staining (assay of cell damage). Kainate induced about a 2.5-fold increase in 5-LO mRNA and triggered a redistribution of 5-LO like immunoreactivity from the pyramidal cell bodies into the dendrites of these neurons, particularly in the CA3 area. The results suggest that glutamate receptor-mediated signaling may modify the expression of neuronal 5-LO and that this enzyme might be involved in glutamte receptor-mediated neuronal plasticity and/or degeneration. Show more

Keywords: leukotriene, kainate, 5-lipoxygenase, inflammation, melatonin, eicosanoids, glutamate, hippocampus

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 81-85, 1998

Authors: Hanbauer, Ingeborg | Galdzicki, Zygmunt | Rapoport, Stanley I. | Scortegagna, Marzia

Article Type: Research Article

Abstract: In the trisomy 16 mouse the increased gene dosage of SOD-1 increases H2O2 production that results in increased oxidative stress. We report here that in hippocampal primary cultures, metallothionein (MT)-I/II immunoreactivity was present mainly in glial fibrillary acidic protein-immunolabeled cells. Western blot analysis showed a two-fold higher level of MT-I/II in trisomy 16 mice then in euploid littermates. In contrast, the immunoreactivity of glutamine synthetase, another glia-expressed protein, was similar in hippocampal …cultures of trisomy 16 mouse and euploid littermates. Oxyblot analysis of hippocampal cultures showed that the carbonyl content in several protein bands was higher in trisomy 16 mice than in euploid littermates giving evidence for increased oxidative stress in trisomy 16 mouse cultures. To evaluate the responsiveness of MT-I/II to agents that increase the level of reactive oxygen species in cells we measured the effect of H2O2, kainic acid, (±) ACPD, and B-amyloid peptide 1-42. Western blot analysis documented that in hippocampal cultures of euploid littermates MT-I/II was maximally increased by 50 µM H2O2, 100 µM kainic acid, 10 µM (±)ACPD, or 1.0 mM B-amyloid peptide 1-42, whereas in those of trisomy 16 mice no furter increase above the elevated level was observed. Our data suggest that in the trisomy 16 mouse the production of reactive oxygen species may have shifted the intracellular redox environment that could have alerted the susceptibility of MT-I/II transcription. The possibilty that transcription factors whose activation may be essential to initiate MT-I/II transcription get oxidized has yet to be examined. Show more

Keywords: trisomy 16 mouse, oxidative stress, metallothionein, antioxidant response, hydrogen peroxide, ionotropic, metabotropic glutamate receptor

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 87-93, 1998

Authors: Scortegagna, Marzia | Chikhale, Elsbeth | Hanbauer, Ingeborg

Article Type: Research Article

Abstract: E14 mesencephalic cultures grown 6 days in Neurobasal Medium containing 10% horse serum consist of differentiated neurons and astroglia. In these cultures, glutathione and metallothionein-I/II are enriched in astrocytes and play an important role in heavy metal scavenging and oxidative stress response. A 24 h exposure to 25 µM Pb, in serum-containing medium, elevated the glutathione content by more than twofold and increased the metallothionein I/II-immunolabeled protein band. In contrast, exposure to 3 to 25 µM …Pb is serum-free medium increased Pb uptake by cells 2 to 4-times above the levels found in 10% serum-containing medium, reduced the glutathione level and obliterated the metallothionein-I/II protein band. The rapid decrease of metallothionein-I/II and glutathione levels in serum-free medium implies that their regulation may depend on a serum factor operative in inducing immediate early genes. Exposure to 6 µM Pb in serum-free or in B27-supplemented medium increased the carbonyl content of several protein bands above control levels indicating that under conditions that curtail metallothionein induction Pb exposure causes increased oxidative stress. Show more

Keywords: primary cultures, lead, glutathione, metallothionein, serum deprivation, oxidative stress

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 95-101, 1998

Authors: Ebadi, M. | Ramana Kumari, M.V. | Hiramatsu, M. | Hao, R. | Pfeiffer, R.F. | Rojas, P.

Article Type: Research Article

Abstract: The finding that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) elicits parkinsonism in human beings suggests that endogenous or xenobiotic neurotoxic compounds may be involved in the etiology of Parkinson's disease (PD). We have shown that cerebrospinal fluid (CSF) of newly diagnosed and drug untreated patients with PD contains a low molecular weight substance(s) which inhibits the growth and function of dopaminergic neurons in culture. In addition, selegiline in a dosage below the level that inhibits monoamine …oxidase B (MAO-B), protects dopaminergic neurons in culture against toxic factor(s) present in the CSF of patients with PD, and the said effect is mediated via elaboration of brain-derived neurotrophic factor (BDNF). In view of the fact that 6-hydroxydopamine (6-OHDA) or MPTP causes parkinsonism by generating free radicals, and inducers of metallothionein (MT) isoforms avert the said neurotoxicity, we intended to learn whether MT isoforms were capable of scavenging free radicals. By employing electron spin resonance spectroscopy (ESR), we examined for the first time the free radical scavenging effects of MT-I and MT-II isoforms on four types of free radicals. Solutions of 0.15 mM of MT-I and 0.3 mM of MT-II scavenged the 1,1-diphenyl-2-picrylhydrazyl radicals completely. Furthermore, they were able to scavenge hydroxyl radicals generated in a Fenton reaction. Moreover, MT-I scavenged almost 90% of the superoxide generated by the hypoxanthine and xanthine oxidase system, while MT-II could only scavenge 40%. By using 2,2,6,6-tetramethyl-4-piperidone as a "spin-trap" for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin, and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidone-1-oxyl, we observed that MT-II could scavenge 92%, while MT-I could completely scavenge all the reactive species generated. The results of this investigation are interpreted to suggest that selegiline by preventing the generation of free radicals, MT isoforms by scavenging free radicals, and neurotrophins by rescuing dopaminergic neurons are capable of attenuating oxidative stress and of providing neuro-protection in PD. Show more

Keywords: selegiline, 6-hydroxydopamine, dopamine, nerve growth factor, brain-derived neurotrophic factor, oxygen free radicals

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 103-111, 1998

Authors: Yu, P.H. | Zhang, X. | Zuo, D.M. | Lai, C.T. | Tieu, K. | Davis, B.A. | Boulton, A.A.

Article Type: Research Article

Abstract: Several clinical investigations have indicated that R-deprenyl, a typical monoamine oxidas B inhibitor, delays the progression of Parkinson's and Alzheimer's disease. A number of aliphatic N-methylpropargylamines, such as R-2-hexyl-N-methylpropargylamines (R-2HxMP), have been found to be highly potent, irreversible, selective, MAO-B inhibitors both in vitro and in vivo. These aliphatic propargylamines do not affect noradrenaline of dopamine uptake and are chemically without an amphetamine moiety and therefore do not exhibit any amphetamine-like effects. …They are capable of protecting mouse striatal dopamine neurons against MPTP-induced toxicity in the caudate, against MK-801-induced apoptosis in the retrosplenial cortex and against DSP-4-induced depletion of naradrenergic axons. They rescue hippocampal neurons in rodents following kainate-induced neuronal damage. They block the expression of heat shock protein (HSP70) and delayed c-Fos expression in hippocampal CA1 region as elicited by kainate. Confocal microscopy also revealed prevention of neuronal damage in hippocampal slices under hypoxia-hypoglycemia conditions. Aliphatic N-methylpropargylamines may be useful in the treatment of neurodegenerative disorders. The mechanism and site of action of the neurorescue effect of these propargylamines, however, remains to be established. Show more

Keywords: MAO, neuroprotection, neurorescue

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 113-118, 1998

Authors: Wood, J.P.M. | Pergande, G. | Osborne, N.N.

Article Type: Research Article

Abstract: We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively …labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 µM - 1 mM) was 100 µM. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production. Show more

Keywords: apoptosis, glutathione, buthionine sulphoxamine, retinal pigmented epithelium, flupirtine gluconate

Citation: Restorative Neurology and Neuroscience, vol. 12, no. 2-3, pp. 119-125, 1998