Aluminum increases levels of beta-amyloid and ubiquitin in neuroblastoma but not in glioma cells

Several epidemiological studies suggest the involvement of aluminum (Al) in the pathogenesis of Alzheimer's disease (AD). There is an increase in the levels of Abeta and ubiquitin in the pathological lesions of AD. Therefore, we have investigated whether aluminum (Al) treatment alters the levels of Abeta and ubiquitin in murine neuroblastoma (NBP2) and rat glioma (C-6) cell cultures. At a low concentration (10 microM), aluminum sulfate stimulated the level of immunoreactive Abeta and ubiquitin in NBP2 cells without changing the levels of the amyloid precursor protein (APP). However, at higher concentrations (100 and 500 microM), aluminum failed to elicit any significant effect on beta-amyloid, whereas ubiquitin levels continued to increase. No changes in the Abeta and ubiquitin content were found in the C-6 glioma cells following treatment with Al at any of the concentrations tested. Exposure of cells to aluminum salts did not alter the rate of proliferation in either of the two cell lines. These data suggest that one of the mechanisms by which Al may play a role in AD is by promoting the formation of Abeta and ubiquitin in neurons.     

D-Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D-glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0-40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time-dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.     

DIFFERENTIATION OF NEUROBLASTOMA, GLIOMA, AND HYBRID CELLS IN CULTURE AS MEASURED BY THE SYNTHESIS OF SPECIFIC PROTEIN SPECIES: EVIDENCE FOR NEUROBLAST-GLIOBLAST RECIPROCAL GENETIC REGULATION

Abstract— Protein species from differentiating neuroblastoma, glioma, and hybrid neuroblastoma-glioma cell lines in cell culture were separated and identified initially in the first dimension by the use of isoelectric focusing gels and were further separated in the second dimension by SDS-acrylamide gels. There were two main classes of proteins identified: proteins which were dominantly expressed in neuroblastoma and also in hybrid cell cultures, and proteins which were expressed in glioma and also hybrid cell cultures. In general, proteins were identified which were significantly expressed in neuroblastoma cells and much reduced in glioma cultures, and also conversely so. The hybrid cell line expressed many of the neuroblastoma-type proteins and relatively fewer of the glioma type proteins. A specific protein species (2) was identified in hybrid cells and was not present in either parental neuroblastoma or glioma cultures. Protein z was expressed however by the co-culturing of neuroblastoma and glioma cells suggesting its induction is dependent on a soluble factor. Protein z in hybrid cells was demonstrated in both stained gels and by autoradiography. Chromosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. It is suggested that similar neuronal-glial interaction may be functional in the intact brain, and that similar reciprocal modulation between neurons and glia may be a central mechanism of differentiation in the nervous system.     

EFFECTS OF ALUMINUM SALTS ON CULTURED NEUROBLASTOMA CELLS

Experimental induction of neurofibrillary tangles was demonstrated several years ago in the central nervous system of rabbits injected with aluminum salts. In the current studies, neuroblastoma cells, capable of morphologic and biochemical differentiation in monolayer culture, have been exposed to medium containing aluminum phosphate; such treatment resulted in an abundant accumulation of 100 Å neurofilaments after 6 days of continous exposure to the aluminum salt. While growth rates and incorporation of radioactive thymidine in treated cells remained similar to controls, total cellular protein, and incorporation of radioactive leucine were significantly increased. Paradoxically, when the protein content of aluminum-treated cultures was maximal, these cultures contained about 20 per cent less ribosomal RNA per cell than in control cultures. In addition, activity of an important neuronal protein, i.e. acetylcholinesterase, was depressed in treated cultures to a level below control values. Both temporal and morphologic similarities between treated neuroblastoma cultures and animals injected with aluminum salts suggest that the observed changes in macromolecular synthesis in cell culture are relevant to in vivo studies.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Ethanol-induced alteration in activities of cerebral phosphatidylinositol 4,5-biphosphate-specific and cytosolic phospholipase C in the brain: analysis using NG 108-15 cells and brains from ethanol-inhaled mice

Effect of long-term exposure to ethanol (EtOH) on the phosphatidylinositol 4,5-biphosphate (PIP₂)-specific and cytosolic phospholipase C (PLC) activities in neuroblastoma x glioma hybrid (NG 108-15) cells and the brains from EtOH-inhaled mice were investigated. Long-term (2 days) exposure of NG 108-15 cells to EtOH induced significant decrease in PIP₂-specific PLC activity dependent on concentration and duration of exposure, although the presence of EtOH in the enzyme assay system induced no alteration in PIP₂-specific PLC activity. On the other hand, cytosolic PLC activity in NG 108-15 cells significantly increased by both the long-term exposure of the cells to EtOH and the addition of EtOH into the assay system. These changes in activities of both types of PLC in NG 108-15 cells observed after EtOH exposure recovered rapidly by the removal of EtOH. Moreover, the changes in activities of PIP₂-specific and cytosolic PLC in the brain of EtOH-inhaled mice were similar to those found in NG 108-15 cells. These results indicate that EtOH inhibits the activity of PIP₂-specific PLC and activates cytosolic PLC in the brain. These changes in cerebral PLC activities are suggested to involve in central action of EtOH and establishment of alcohol dependence.     

Metals accelerate production of the aberrant splicing isoform of the presenilin-2

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.     

Differences in the metabolism of inositol and phosphoinositides by cultured cells of neuronal and glial origin

Phosphoinositide and inositol metabolism was compared in glioma (C6), neuroblastoma (N1E-115) and neuroblastoma X glioma hybrid (NG 108-15) cells. All cell lines had similar proportions of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Neuroblastoma and hybrid cells had almost identical phospholipid and phosphoinositide compositions and similar activities for the enzymes metabolizing polyphosphoinositides (PI kinase, PIP phosphatase, PIP kinase, PIP2 phosphatase, PIP2 phosphodiesterase). Glioma cells differed by having greater proportions of ethanolamine plasmalogen and sphingomyelin, lower PIP kinase, 3-5-fold higher PIP phosphatase activity and 10-15-fold greater PIP2 phosphodiesterase activity. Higher PIP phosphatase and PIP2 diesterase activities appear to be characteristic of cells of glial origin, since similar activities were found in primary cultures of astroglia. Glioma cells also metabolize inositol differently. In pulse and pulse-chase experiments, glioma cells transported inositol into a much larger water-soluble intracellular pool and maintained a concentration gradient 30-times greater than neuroblastoma cells. Label in intracellular inositol was less than in phosphoinositides in neuroblastoma and exchanged rapidly with extracellular inositol. In glioma, labeling of intracellular inositol greatly exceeded that of phosphoinositides. As a consequence, radioactivity in prelabeled phosphoinositides could not be effectively chased from glioma cells by excess unlabeled inositol. Such differences between cells of neuronal and glial origin suggest different and possibly supportive roles for these two cell types in maintaining functions regulated through phosphoinositide-linked signalling systems in the central nervous system.     

低氧对NG108－15细胞生长及分化的影响

神经母细胞瘤和神经胶质瘤细胞融合的克隆细胞系ＮＧ１０８－１５细胞在含分化剂双丁基环化单磷酸腺苷（ｄＢｆｃＡＭＰ）的培养液培养后分化,成为具神经细胞特征的细胞。本实验利用四唑盐（ＭＴＴ）微量比色法,并结合焦油紫染色,测定和观察细胞的生长及分化状况,研究了低氧（２％Ｏ２＋９３％Ｎ２＋５／ＣＯ２）对未分化的,分化中的和已分化完成的ＮＧ１０８－１５细胞的影响。获得的主要结果是;低氧明显降低未分化细胞增殖和存活率,使分化完成的细胞大量死亡;低氧影响ＮＧ１０８－１５细胞的分化,使细胞在分化中出现体积膨大,突起短等异常特征,经焦油紫染色,胞质中无尼氏体（即不着色）的细胞增多。低氧是否可能使未分化ＮＧ１０８－１５细胞向更多地表达胶质细胞特征的方向分化？将是一个十分有趣的问题。     

Glycolytic metabolism in cultured cells of the nervous system

The effects of thiamine deficiency and of the antithiamine drug pyrithiamine on the C-6 glioma and the C-1300 neuroblastoma cell lines have been studied. Thiamine deficiency increased the doubling time of the neuroblastoma cells without affecting that of the glioma cells. Pyrithiamine prevented both cell lines from doubling even once. (hiamine deficiency had only slight effects on intracellular pyruvate and lactate levels or on efflux rates for the acids, but pyrithiamine treatment resulted in large increases in both the intracellular levels and the efflux in both cell lines. For comparison, the pyruvate and lactate levels in mouse brain were measured. The levels from thiamine-deficient mouse brain were essentially unchanged from controls while pyrithiamine treatment caused a significant elevation only of the pyruvate concentration.     

A model of aluminum exposure associated with lipid peroxidation in rat brain

We have developed a rat model to investigate the relationship between aluminum exposure and aluminum accumulation, and with oxidative damage in brain tissues. Intraperitoneal injections of aluminum lactate for 7 wk (the total aluminum dosage per rat was approx 100 mg) significantly increased aluminum levels in the brain. The concentration of lipid peroxidation products (thiobarbituric acid-reactive substances [TBARS]) also increased in the brain following aluminum lactate injections. No significant correlations between the concentrations of aluminum and of TBARS were found in the whole brain. Subcellular analysis revealed that aluminum lactate injections led to a significant increase in the concentration of aluminum in the mitochondrial fraction but had no significant effect on the concentration of peroxides in any subcellular fraction. These results suggest that aluminum accumulation induced by the aluminum lactate administration associates with the acceleration of lipid peroxidation in rat brain. Furthermore, these data indicate that the pro-oxidant effect of aluminum may be indirect and concentration independent. The experimental conditions used here provide an animal model of aluminum accumulation in the brain that should prove useful for further investigations of the mechanisms of aluminum neurotoxicity.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

α-Aryl-N-aryl nitrones: Synthesis and screening of a new scaffold for cellular protection against an oxidative toxic stimulus

Nitrone-containing compounds are commonly employed as spin traps of free radical species in chemical and biological studies. Some molecules as α-phenyl-N-t-butyl nitrone (PBN) and its derivatives have been tested as potential drugs to treat oxidative stress related diseases, as Alzheimer and stroke for example. In this work we report the design and the synthesis of α-aryl-N-aryl nitrones and their cytoprotection profile on human neuroblastoma cells (SH-SY5Y) under induced oxidative stress. All the nine synthesized nitrones showed a significant response at low micromolar concentration. The selected compound 8 (α-phenyl-N-phenyl nitrone) increased the reduced glutathione (GSH) levels by 65% and lowered the necrotic cell death from 25.8% to 3.8%. Based on our data, the designed highly conjugated nitrone double-bond skeleton can be considered as a good scaffold for further studies regarding oxidative stress-related diseases.     

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.     

Glycolytic metabolism in cultured cells of the nervous system

Pyruvate and lactate efflux from C-6 glioma cells has been found to be regulated by both the medium glucose concentration and the medium concentration of the two acids. Each moves down a concentration gradient until the extracellular level is in equilibrium with the intracellular. Long-term growth studies demonstrated that the cells preferentially utilize glucose but that once it is depleted, they will take up first pyruvate, followed by lactate, for further metabolism. Changes in the intracellular levels of the two metabolites correspond to those seen in the medium. The rate of glycogen breakdown parallels that of medium glucose ultilization. Preliminary results with the C-1300 neuroblastoma cells showed pyruvate and lactate efflux rates comparable to those of the glioma cells.     

Down-regulation of immediate early gene egr-1 expression in rat C6 glioma cells by short-term exposure to high salt culture medium

Influence of high salt culture conditions on the expression of immediate early gene egr-1 in rat C6 glioma cells was investigated by measuring both Egr-1 mRNA and protein levels in the cells exposed to the medium containing high concentrations of NaCl. The exposure to high salt medium reduced Egr-1 mRNA and protein levels, while Egr-1 mRNA levels were not altered by the medium containing either sucrose or glycerol. Veratridine and monensin also reduced Egr-1 mRNA levels, similar in extent to that induced by high salt medium. Imaging analysis indicated that the exposure to high salt medium induced the elevation of Na+ levels within the cells. These results indicate that neither hyperosmotic pressure nor ionic strength of high salt medium contribute to the reduction of Egr-1 expression, and suggest that the elevation of intracellular Na+ concentration is closely associated with the down-regulation of egr-1 gene expression.     

Effect of lycopene on As2O3 induced oxidative stress in SH-SY5Y cells

It is known that oxidative stress may cause neuronal injury and several experimental models showed that As₂O₃ exposure causes oxidative stress. Lycopene, a carotenoid, has been shown to have protective effect in neurological disease models due to antioxidant activity, but its effect on As₂O₃-induced neurotoxicity is not identified yet. The aim of this study is to investigate the effects of lycopene on As₂O₃-induced neuronal damage and the related mechanisms. Cell viability was determined by the MTT assay. Lycopene was administrated with different concentrations (2, 4, 6 and 8 µM) one hour before 2 µM As₂O₃ exposure in SH-SY5Y human neuroblastoma cells. The anti-oxidant effect of lycopene was determined by measuring superoxide dismutase (SOD), catalase (CAT) hydrogen peroxide (H₂O₂), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS). MTT results and LDH cytotoxicity analyses showed that pretreatment with 8 µM lycopene significantly improved the toxicity due to As₂O₃ exposure in SH?SY5Y neuroblastoma cells. Pretreatment with lycopene significantly increased the activities of anti?oxidative enzymes as well as total antioxidant status and decreased total oxidative status in As₂O₃ exposed cells. The results of this study indicate that lycopene may be a potent neuroprotective against oxidative stress and could be used to prevent neuronal injury or death in several neurological diseases.

     

Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H(2)O(2)

Glioblastomas are notorious for their resistance to ionizing radiation and chemotherapy. We hypothesize that this resistance to ionizing radiation is due, in part, to alterations in antioxidant enzymes. Here, we show that rat and human glioma cells overexpress the antioxidant enzyme peroxiredoxin II (Prx II). Glioma cells in which Prx II is decreased using shRNA exhibit increased hyperoxidation of the remaining cellular Prxs, suggesting that the redox environment is more oxidizing. Of interest, decreasing Prx II does not alter other antioxidant enzymes (i.e., catalase, GPx, Prx I, Prx III, CuZnSOD, and MnSOD). Analysis of the redox environment revealed that decreasing Prx II increased intracellular reactive oxygen species in 36B10 cells; extracellular levels of H(2)O(2) were also increased in both C6 and 36B10 cells. Treatment with H(2)O(2) led to a further elevation in intracellular reactive oxygen species in cells where Prx II was decreased. Decreasing Prx II expression in glioma cells also reduced clonogenic cell survival following exposure to ionizing radiation and H(2)O(2). Furthermore, lowering Prx II expression decreased intracellular glutathione and resulted in a significant decline in glutathione reductase activity, suggesting a possible mechanism for the observed increased sensitivity to oxidative insults. Additionally, decreasing Prx II expression increased cell cycle doubling times, with fewer cells distributed to S phase in C6 glioma cells and more cells redistributed to the most radiosensitive phase of the cell cycle, G2/M, in 36B10 glioma cells. These findings support the hypothesis that inhibiting Prx II sensitizes glioma cells to oxidative stress, presenting Prxs as potential therapeutic targets.     

Neuroprotective effect of carvedilol and melatonin on 3-nitropropionic acid-induced neurotoxicity in neuroblastoma

In neurodegenerative diseases, progressive oxidative stress is a major event that precedes neuronal death. Oxidative stress is characterized by an imbalance between oxidants and antioxidants. This imbalance induced oxidative molecular and cell damage, reducing cellular viability. 3-Nitropropionic acid (3NP) causes oxidative stress and other molecular and cellular changes similar to those observed in neurons of patients with Huntington’s disease. Since carvedilol and melatonin act as free-radical scavengers, this study examined the effect of carvedilol (10^?5 M) and melatonin (10^?5 M) on oxidative and cell damage induced by 3NP in N1E-115 neuroblastoma cells. Carvedilol and melatonin prevented the increases in lipid peroxidation and total LDH activity, as well as the depletion of reduced glutathione (GSH) and the reduction of antioxidative enzymes activities in N1E-115 cells incubated with 100 mM 3NP. All these carvedilol and melatonin effects were more intense when the drugs were added before rather than after inducing the damage by 3NP. These results also provided evidence supporting the hypothesis that carvedilol and melatonin can be useful for treating neurodegenerative diseases, such as Huntington’s disease.