4-hydroxy-2-nonenal and Neuroblastoma

9,10-Phenanthrenequinone (PQ), a major quinone component in diesel exhaust particles, is considered to provoke damage of respiratory and vascular cells through highly producing reactive oxygen species (ROS), but little is known about its pathophysiological role in neuronal cell damage. In this study, we found that incubation with 1,2-naphthoquinone, 1,4-naphthoquinone and PQ, major quinone components in diesel exhausts, provokes apoptosis of human neuroblastoma cell lines. SK-N-SH cell treatment with a lethal concentration of PQ facilitated ROS production within 6 h. The treatment also promoted formation of 8-hydroxy-deoxyguanosine, p53 activation, elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, and resultant activation of caspase-9 and caspase-3, inferring that ROS production, DNA damage and mitochondrial dysfunction are crucial processes of the PQ-triggered SK-N-SH cell apoptosis. The PQ treatment of SK-N-SH cells elevated the level of 4-hydroxynonenal (HNE), a cytotoxic reactive aldehyde generated from lipid peroxidation. The treatment with PQ and HNE also decreased cellular levels of total and reduced glutathiones, and the damage elicited by HNE was ameliorated and deteriorated by pretreating with cell-permeable glutathione analog and the depletor, respectively. Moreover, the treatment with PQ and HNE decreased the proteasomal proteolytic activities, suggesting a contribution of decrease in the antioxidant abilities to the ROS-mediated neuroblastoma cell apoptosis. Our comparative analyses of 17 cells showed a positive correlation between the PQ reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) activities. In addition, overexpression and knockdown of NQO1 augmented and lowered, respectively, the ROS production through PQ redox-cycling and the quinone toxicity. Furthermore, the treatment with PQ and HNE up-regulated the NQO1 expression. Taken together, PQ exposure produces large amounts of ROS in neuroblastoma cells via NQO1 up-regulation and resultant acceleration of its redox-cycling, followed by activation of the ROS-dependent apoptotic mechanism.

Topics: Air Pollutants; Aldehydes; Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Glutathione; Humans; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); Neuroblastoma; Neurons; Phenanthrenes; Proteasome Endopeptidase Complex; Reactive Oxygen Species

Activity of neprilysin (NEP), the major protease which cleaves amyloid-β peptide (Aβ), is reportedly reduced in the brains of patients with Alzheimer's disease (AD). Accumulation of Aβ generates reactive oxygen species (ROS) such as 4-hydroxynonenal (HNE), and then reduces activities of Aβ-degrading enzymes including NEP. Xanthorrhizol (Xan), a natural sesquiterpenoid, has been reported to possess antioxidant and anti-inflammatory properties. The present study examined the effects of Xan on HNE- or oligomeric Aβ

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Brain; Cell Line; Humans; Neprilysin; Neuroblastoma; Neuroprotective Agents; Oxidation-Reduction; Oxidative Stress; Peptide Fragments; Phenols; Reactive Oxygen Species

4-hydroxy-2-nonenal (HNE), a toxic lipid peroxidation product, is associated with oxidative damage in cells and involved in various diseases including the initiation and progression of cancer. Cancer cells have a high, adaptable metabolism with a shift from oxidative phosphorylation to glycolysis and rely on high levels of glucose and glutamine as essential nutrients for cell growth. Here we investigated whether the toxic effects of HNE on the mitochondrial membrane potential (MMP) of cancer cells depends on their metabolic state by deprivation of glucose and/or glutamine. The addition of 16 μM HNE to N18TG2 neuroblastoma cells incubated in glucose medium led to a severe reduction of MMP, which was similar to the MMP of cells fed with both glucose and glutamine. In contrast, HNE addition to cells starved in glutamine medium increased their MMP slightly for a prolonged time period and this was accompanied by increased cellular survival. We found that ß-oxidation of HNE did not cause the increased MMP, since the aldehyde dehydrogenase was distinctly more active in cells with glucose medium. However, after blocking fatty acid ß-oxidation in cells starved in glutamine medium with etomoxir, which inhibits carnitine palmitoyltransferase 1, HNE addition induced a strong reduction of MMP similar to cells in glucose medium. Surprisingly, the effect of more toxic 4-oxo-2-nonenal was less pronounced. Our results suggest that in contrast to cells fed with glucose, glutamine-fed cancer cells are capable of ß-oxidizing fatty acids to maintain their MMP to combat the toxic effects of HNE.

Topics: Aldehydes; Animals; Cell Line, Tumor; Cell Shape; Cell Survival; Energy Metabolism; Glucose; Glutamine; Lipid Peroxidation; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroblastoma; Oxidation-Reduction; Oxidative Stress; Time Factors

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.

Topics: Air Pollutants; Aldehydes; Analysis of Variance; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Electrophoretic Mobility Shift Assay; Humans; Hydrogen Sulfide; Lipid Peroxidation; Neuroblastoma

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell viability, modifications of NO, intracellular Ca(2+) concentration [Ca(2+)](i) and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca(2+)](i) immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O(2) hypoxia induces variations of NO and [Ca(2+)](i) with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.

Topics: Adenosine Triphosphate; Aldehydes; Calcium; Cell Hypoxia; Cell Line, Tumor; Cell Survival; F2-Isoprostanes; Humans; Iron; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Nitric Oxide; Oxygen; Protein Carbonylation

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

Topics: Acrolein; Aldehydes; Analysis of Variance; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytotoxins; Flavonoids; Free Radicals; Glutathione; Humans; Lipid Peroxidation; Luminescence; NADPH Oxidases; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Plant Extracts; Protein Carbonylation; Tyrosine

In Alzheimer's disease (AD), oxidative stress-induced lipid peroxidation leads to accumulation of unsaturated aldehydes including acrolein and 4-hydroxynonenal (4HNE) in brain. In this study, we examined the effects of these lipid peroxidation products on apoptotic pathways in cultured neurons. Acrolein and 4HNE increased the levels of active phosphorylated forms of c-jun and CREB, the transcription factors that promote apoptosis and cell survival, respectively. However, they decreased the activity of CREB-dependent BDNF promoter while they increased the activity of promoters responsive to c-jun. We hypothesized that this differential regulation could be due to competition between proapoptotic c-jun and cytoprotective CREB for CBP (CREB-binding protein), a coactivator shared by several transcription factors. In support of this hypothesis, we demonstrate that the decrease of BDNF promoter activity by acrolein and 4HNE could be restored (i) by cotransfection with CBP, (ii) by cotransfection with VP 16-CREB, a constitutively active form of CREB that does not depend on CBP for its activation, or (iii) by inhibiting JNK-mediated c-jun activation. Finally, adenoviral transduction of hippocampal neurons with VP 16-CREB resulted in significant reduction in caspase-3 activation by acrolein and 4HNE. These observations suggest that lipid peroxidation-induced differential regulation of CREB and c-jun might play a role in neurodegeneration in AD.

Topics: Acrolein; Adenoviridae; Aldehydes; Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Caspase 3; Caspases; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Female; Hippocampus; Humans; Lipid Peroxidation; Neuroblastoma; Neurons; Phosphorylation; Pregnancy; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.

Topics: Aldehydes; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Flow Cytometry; Genes, Tumor Suppressor; HL-60 Cells; Humans; Lipid Metabolism; Microscopy, Fluorescence; Neuroblastoma; Nuclear Proteins; Peroxides; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Trans-Activators; Transcription Factors; Tretinoin; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

We have previously demonstrated that neuronal microtubules are exquisitely sensitive to the lipid peroxidation product 4-hydroxynonenal (HNE). The mechanism, however, by which HNE disrupts the microtubules, is not known. Sulfhydryl groups of protein-cysteines constitute main targets of HNE. Indeed, HNE is mainly detoxified by conjugation to glutathione (GSH), a reaction that leads to depletion of cellular GSH. GSH maintains protein sulfhydryl groups in the reduced form and has been implicated in the regulation of cytoskeletal function. Here, we assess what role depletion of cellular GSH plays in the HNE-induced microtubule disruption. We demonstrate that HNE and its intracellularly activated tri-ester analog, HNE(Ac)(3), cause substantial GSH depletion in Neuro2A cells. However, other compounds inducing GSH depletion had no effect on the microtubule network. Therefore, HNE-induced depletion of cellular GSH does not contribute to the HNE-induced microtubule disruption. We previously demonstrated that another main cellular target of HNE is tubulin, the core protein of microtubules containing abundant cysteines. The functional relevance of this adduction, however, had not been evaluated. Here, we demonstrate that exposure of Neuro 2A cells to HNE or HNE(Ac)(3) results in the inhibition of cytosolic taxol-induced tubulin polymerization. These and our previous observations strongly support the hypothesis that HNE-adduction to tubulin is the primary mechanism involved in the HNE-induced loss of the highly dynamic neuronal microtubule network.

Topics: Aldehydes; Cell Line, Tumor; Glutathione; Glutathione Transferase; Growth Inhibitors; Humans; Immunohistochemistry; Microtubules; Neuroblastoma; Neurons; Tubulin

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.

Topics: Aldehydes; Cell Line, Tumor; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lipid Peroxidation; Mitogen-Activated Protein Kinase 1; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction

Inhibition of the proteasomal pathway for degrading abnormal proteins leads to protein aggregation, increased oxidative damage and increased protein nitration. We now show that interference with polyubiquitination has similar consequences. Expression of a dominant-negative mutant form of ubiquitin (K48R) in NT-2 and SK-N-MC cells caused decreased cell growth rates and increased oxidative damage (protein carbonyls and lipid peroxidation), nitric oxide production and elevated protein nitration. It also rendered cells highly sensitive to 4-hydroxy-2,3-trans-nonenal, a neurotoxic end-product of lipid peroxidation, hydrogen peroxide and deprivation of growth factors. Overexpression of wild-type ubiquitin did not produce these effects. Our data show that interference with the ubiquitin-proteasome pathway at a different point and by a different mechanism can produce many of the common features of human neurodegenerative diseases, such as increased lipid peroxidation, protein oxidation and protein nitration. We suggest that defects in this pathway at multiple points could produce the common features of neurodegenerative diseases, and that more such defects remain to be discovered.

Topics: Aldehydes; Antioxidants; Cell Division; Cell Line; Cell Survival; Clone Cells; Cysteine Endopeptidases; Enzyme Inhibitors; Genes, Dominant; Glutathione; Green Fluorescent Proteins; Humans; Luminescent Proteins; Multienzyme Complexes; Mutation; Neuroblastoma; Neurodegenerative Diseases; Nitrates; Nitrites; Oxidants; Oxidative Stress; Proteasome Endopeptidase Complex; Proteins; Recombinant Fusion Proteins; Teratocarcinoma; Transfection; Ubiquitins

Cdk5, a member of the cyclin-dependent kinase (cdk) family, is predominantly active in neurons, where its activity is tightly regulated by the binding of its neuronal activators p35 and p39. Cdk5 is implicated in regulating the proper neuronal function; a deregulation of cdk5 has been found associated with Alzheimer's disease and amyotrophic lateral sclerosis. As oxidative stress products have been seen co-localized with pathological hallmarks of neurodegenerative diseases, we studied the effect of oxidative stress on the cdk5 enzyme in human neuroblastoma IMR-32 cells. We evaluated the effects of 4-hydroxynonenal and Ascorbate plus FeSO(4) on cdk5 activity and on the expression of cdk5 and p35 proteins. We report here that oxidative stress stimulates cdk5 activity and induces an upregulation of its regulatory and catalytic subunit expression in IMR-32 vital cells, showing that the cdk5 enzyme is involved in the signaling pathway activated by oxidative stress.

Topics: Aldehydes; Alzheimer Disease; Amyotrophic Lateral Sclerosis; Cell Survival; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Enzyme Activation; Ferrous Compounds; Humans; Microscopy, Phase-Contrast; Nerve Tissue Proteins; Neuroblastoma; Oxidative Stress; Signal Transduction; Tumor Cells, Cultured; Up-Regulation

4-hydroxy-trans-2-nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4-hydroxy-trans-2-nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non-toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time-dependent and dose-dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 micro m HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.

Topics: Aldehydes; Animals; Biomarkers; Cell Line; Cerebral Cortex; Dose-Response Relationship, Drug; Hydroxy Acids; In Vitro Techniques; Male; Mitochondria; Neuroblastoma; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Subcellular Fractions

4-Hydroxy-2,3-trans-nonenal (HNE) is a neurotoxic unsaturated aldehyde end-product of lipid peroxidation. The addition of HNE to NT-2 and SK-N-MC cell lines induces apoptosis and we now investigated the time-course of events occurring prior to apoptosis. Treatment of both NT-2 and SK-N-MC cell lines with HNE led to HNE association with the proteasome, increased levels of protein carbonyls and ubiquitinated proteins, and decreased proteasomal function. There was also decreased metabolic activity, cytochrome c release and activation of caspase 3, followed by apoptotic changes including chromatin condensation, cell shrinkage and DNA fragmentation and laddering. Overexpression of mutant superoxide dismutase 1 proteins associated with amyotrophic lateral sclerosis decreased proteasomal activities in the absence of HNE and accelerated the apoptosis induced by HNE. By contrast, overexpression of wild-type superoxide dismutase 1 did not affect basal levels of proteasomal activity. The data suggest that accumulation of ubiquitinated proteins and impairment of proteasomal function are important events in HNE toxicity. We propose that the proteasomal system is a significant target of HNE neurotoxicity in a wide range of neurodegenerative diseases, especially if abnormal proteins are being expressed.

Topics: Aldehydes; Apoptosis; Caspase 3; Caspases; Cell Membrane; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Lipid Peroxidation; Multienzyme Complexes; Mutation; Neuroblastoma; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Superoxide Dismutase; Teratocarcinoma; Tumor Cells, Cultured; Ubiquitin

4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation and has been suggested to play a role in the pathogenesis in several common diseases as well as injuries caused by environmental toxicants. Although formed intracellularly in vivo, for practical reasons this molecule is applied extracellularly in order to analyze its biological effects. The focus of this study was to develop an approach that would enable intracellular HNE production in the absence of the many other products and processes that occur in cells experiencing generalized oxidative stress. To this end, we synthesized 1,1,4-tris(acetyloxy)-2(E)-nonene (HNE[Ac]3), a triester analogue of HNE that is itself unreactive but could be hydrolyzed intracellularly presumably by lipases and/or esterases into the highly reactive HNE. In vitro lipase rapidly converted HNE(Ac)(3) initially to 4-acetyloxy-2-nonenal (HNE[Ac]1) and then to HNE. Neuro 2A cell lysate also caused a rapid hydrolysis of HNE(Ac)3 into HNE(Ac)1 and HNE. Incubation of BSA with HNE(Ac)3 resulted in protein-adduct formation only in the presence of lipase. We demonstrated adduction of HNE to proteins in Neuro 2A cells exposed to HNE(Ac)3 by immunoblotting and immunocytochemistry using antibodies specific for HNE-Michael adducts on proteins. We have previously shown that microtubule organization is very sensitive to HNE. Analysis of Neuro 2A cell microtubules showed that this cytoplasmic organelle is similarly sensitive to HNE and HNE(Ac)3.

Topics: Acetates; Aldehydes; Alkenes; Animals; Chromatography, Gas; Fluorescent Antibody Technique, Indirect; Immunoblotting; Lipase; Mice; Microtubules; Neuroblastoma; Proteins; Tumor Cells, Cultured

Here we show, for the first time, the in vitro formation of filamentous aggregates of phosphorylated tau protein in SH-SY5Y human neuroblastoma cells. The formation of such aberrant aggregates, similar to those occurring in vivo in Alzheimer's disease and other tauopathies, requires okadaic acid, a phosphatase inhibitor, to increase the level of phosphorylated tau, and hydroxynonenal, a product of oxidative stress that selectively adducts and modifies phosphorylated tau. Our findings suggest that both phosphorylation and oxidative modification are required for tau filament formation. Importantly, the in vitro formation of intracellular tau aggregates could be used as a model of tau polymerization and facilitate the development of novel therapeutic approaches.

Topics: Aldehydes; Humans; Neuroblastoma; Okadaic Acid; Phosphorylation; Polymers; tau Proteins; Tumor Cells, Cultured

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Topics: 1-Methyl-4-phenylpyridinium; Aldehydes; alpha-Synuclein; Cell Division; Cell Line; Cell Survival; Clone Cells; Culture Media, Serum-Free; Enzyme Inhibitors; Gene Expression; Glutathione; Guanine; Humans; Hydrogen Peroxide; Ketones; Lipid Peroxidation; Mitochondria; Mutation; Nerve Tissue Proteins; Neuroblastoma; Oxidants; Oxidative Stress; Parkinsonian Disorders; Synucleins; Teratocarcinoma; Transfection; Tyrosine

Glutathione-S-transferases (GSTs) are a superfamily of enzymes that function to catalyze the nucleophilic attack of glutathione on electrophilic groups of a second substrate. GSTs are present in many organs and have been implicated in the detoxification of endogenous alpha, beta unsaturated aldehydes, including 4-hydroxynonenal (HNE). Exogenous GST protects hippocampal neurons against HNE in culture. To test the hypothesis that overexpression of GST in cells would increase resistance to exogenous or endogenous HNE induced by oxidative stress, stable transfectants of SY5Y neuroblastoma cells with GST were established. Stable GST transfectants demonstrated enzyme activities 13.7 times (Clone 1) and 30 times (Clone 2) higher than cells transfected with vector alone. GST transfectants (both Clones 1 and 2) demonstrated significantly (p <.05) increased resistance to ferrous sulfate/hydrogen peroxide (20.9% for Clone 1; 46.5% for Clone 2), amyloid beta-peptide (12.2% for Clone 1; 27.5.% for Clone 2), and peroxynitrite (24.3% for Clone 1; 43.9% for Clone 2), but not to exogenous application of HNE in culture medium. GST transfectants treated with 1,1,4-tris (acetyloxy)nonane, a nontoxic derivative of HNE that is degraded to HNE intracellularly, demonstrated a statistically significant (p <.05) increase in viability in a dose-dependent manner compared with SY5Y cells transfected with vector alone. These results suggest that overexpression of GST increases resistance to endogenous HNE induced by oxidative stress or released in the degradation of 1,1,4-tris (acetyloxy)nonane, but not to exogenous application of HNE.

Topics: Aldehydes; Amyloid beta-Peptides; Blotting, Western; Cell Survival; Drug Resistance, Neoplasm; Ferrous Compounds; Gene Expression; Glutathione; Glutathione Transferase; Humans; Hydrogen Peroxide; Isoenzymes; L-Lactate Dehydrogenase; Neuroblastoma; Oxidative Stress; Tetrazolium Salts; Thiazoles; Transfection; Tumor Cells, Cultured

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.

Topics: Aldehydes; Amino Acid Substitution; Cell Death; Cell Survival; Cross-Linking Reagents; Culture Media, Serum-Free; Genes, bcl-2; Guanine; Humans; Hydrogen Peroxide; Kinetics; Lipid Peroxidation; Motor Neuron Disease; Mutagenesis, Site-Directed; Neuroblastoma; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Teratocarcinoma; Tumor Cells, Cultured; Tyrosine

Neuron death and neuron degeneration occur in the CNS during the course of aging. Although multiple cellular alterations transpire during the aging process, those that mediate age-associated neuron death have not been identified. Recent evidence implicates oxidative stress as a possible means of neuron death and neuron degeneration during aging. In the present study, we demonstrate a marked decrease in multicatalytic proteasome activity in the spinal cord of Fisher 344 rats at 12, 24 and 28 months, compared with spinal cord tissue from 3-week- and 3-month-old animals. Application of oxidative injury (FeSO(4)) or the lipid peroxidation product 4-hydroxynonenal decreases multicatalytic proteasome activity in a time- and dose-dependent manner in a motor neuron cell line. Loss of multicatalytic proteasome activity occurs before the loss of multicatalytic proteasome immunoreactivity, with FeSO(4)- and 4-hydroxynonenal-mediated decreases ameliorated by the application of a cell permeable form of the antioxidant glutathione. Application of multicatalytic proteasome inhibitors, but not inhibitors of lysosomal proteases, induced neuron death that was attenuated by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone or N-acetyl-Asp-Glu-Val-Asp-Cho (aldehyde). Together, these data suggest that multicatalytic proteasome inhibition occurs during aging of the spinal cord, possibly as the result of oxidative stress, and that multicatalytic proteasome inhibition may be causally related to neuron death.

Topics: Acetylcysteine; Aging; Aldehydes; Amino Acid Chloromethyl Ketones; Animals; Cell Death; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Glutathione; Iron; Lipid Peroxidation; Lysosomes; Mice; Motor Neurons; Multienzyme Complexes; Neuroblastoma; Oligopeptides; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Inbred F344; Reactive Oxygen Species; Spinal Cord; Tumor Cells, Cultured

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.

Topics: Actin Cytoskeleton; Aldehydes; Animals; Cell Survival; Lipid Peroxidation; Microtubules; Neurites; Neuroblastoma; Neurons; Oxidative Stress; Tubulin; Tumor Cells, Cultured

6-Hydroxydopamine(6-OHDA) and Merocyanine-540(MC-540) have been used clinically for purging of neuroblastoma cells prior to autologous bone marrow transplantation. Both substances were found to be more toxic against neuroblastoma cells than against hematopoietic stem cells. The more pronounced cytotoxic effects of 6-OHDA against neuroblastoma cells were not caused by its selective uptake; the rapid autooxidation at physiological pH leads to the formation of H2O2 already in the incubation medium. Cytotoxic effects were not detected in short-time test systems (4 hour chromium-51 release assay) but only after longer incubation periods. In contrast, MC-540 proved to be toxic almost equally in short- and long-time test systems. 4-Hydroxynonenal(4-HNE) that may be formed in the plasma membrane subsequently to photoactivation of MC-540 was only slightly more toxic to neuroblastoma cells than to hematopoietic cells. Although the use of 6-OHDA and MC-540 in bone marrow purging has some limitations, the sensitivity of neuroblastoma cells against reactive oxygen compounds may be exploited more generally for therapy of this tumor.

Topics: Aldehydes; Antineoplastic Agents; Cell Survival; Hematopoietic Stem Cells; Humans; Hydrogen Peroxide; Hydroxydopamines; Neuroblastoma; Oxidopamine; Pyrimidinones; Tumor Cells, Cultured