okadaic-acid and Neuroblastoma

Topics: Alzheimer Disease; Drugs, Chinese Herbal; Humans; Hydrogen Peroxide; Molecular Docking Simulation; Neuroblastoma; Okadaic Acid

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss, and behavioral disturbances, ultimately resulting in death. The critical roles of glycogen synthase kinase-3β (GSK-3β) in tau pathology have also received considerable attention. Based on molecular docking studies, a series of novel α-carboline derivatives were designed, synthesized, and evaluated as GSK-3β inhibitors for their various biological activities. Among them, compound ZCH-9 showed the most potent inhibitory activity against GSK-3β, with an IC

Topics: Alzheimer Disease; Carbolines; Glycogen Synthase Kinase 3 beta; Humans; Molecular Docking Simulation; Neuroblastoma; Neurodegenerative Diseases; Okadaic Acid; Phosphorylation; Structure-Activity Relationship; tau Proteins

Mycotoxins are emerging toxins in the marine environment, which can co-occur with algal toxins to exert synergistic or antagonistic effects for human seafood consumption. The current study assesses the cytotoxicity of the algal toxin okadaic acid, shellfish, and dust storm-associated mycotoxins alone or in combination on human intestinal (HT-29) and neuroblastoma (SH-SY5Y) cell lines. Based on calculated IC

Topics: Cell Line, Tumor; Cell Survival; Drug Interactions; Humans; Intestines; Mycotoxins; Neuroblastoma; Okadaic Acid

An accumulation of hyperphosphorylated tau in the brain is a hallmark of Alzheimer's disease (AD). Deficits in protein phosphatase 2A (PP2A) are associated with tau hyperphosphorylation in AD.. To investigate the effects of morroniside (MOR), isolated from Cornus officinalis, on tau hyperphosphorylation and its underlying mechanisms related to PP2A.. SK-N-SH cells were pretreated with 50-200 μM MOR for 24 h followed by 20 nM okadaic acid (OA) for 6 h. PP2Ac siRNA was transfected into HEK293 cells to determine the direct interaction of MOR with PP2A. Western blotting was used to measure the expression of proteins and enzymes. PP2A activity was measured by molybdenum blue spectrophotometry.. Pretreatment with MOR improved the cellular morphological damage and inhibited tau hyperphosphorylation in SK-N-SH cells induced by OA, a PP2A inhibitor. Moreover, MOR increased PP2A activity, concurrent with a decrease in the expression of demethylated PP2A at Leu309 and phosphorylated PP2A at Tyr307. MOR decreased protein phosphatase methylesterase 1 (PME-1) expression and the ratio of PME-1/leucine carboxyl methyltransferase 1 (LCMT-1). Furthermore, MOR treatment decreased the phosphorylation of Src at Tyr416, which regulates the phosphorylation of PP2A. MOR had no effect on PP2Ac expression and tau hyperphosphorylation in PP2Ac siRNA-transfected cells.. MOR attenuated OA-induced tau hyperphosphorylation via PP2A activation, and its mechanism might be related to the regulation of PP2Ac post-translational modification and upstream enzymes such as Src and PME-1.

Topics: Analysis of Variance; Cell Line; Enzyme Inhibitors; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Glycosides; HEK293 Cells; Humans; Neuroblastoma; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; RNA, Small Interfering; tau Proteins; Transfection; Tyrosine

Neurofibrillary tangles (NFTs), which are composed of intracellular filamentous aggregates of hyperphosphorylated tau protein, are one of the pathological hallmarks of Alzheimer's disease (AD). Because tau phosphorylation is regulated by phosphatases, abnormal metabolism of protein phosphatase 2A (PP2A) has been proposed to be a contributing factor to the disease process.. To determine the function of folic acid on tau phosphorylation, an in vitro model of human neuroblastoma cells (SH-SY5Y) were exposed to folic acid (0-40 μmol/L) for 96 h, in the presence or absence of the phosphoesterase inhibitor okadaic acid (OA) (10 nmol/L) for 9 h. The data of western blot showed tau phosphorylation at the Ser396 site in OA-incubated SH-SY5Y cells was inhibited by folic acid in a concentration-dependent manner, with the folic acid concentration of 40 μmol/L providing maximal inhibition. Folic acid can downregulate tau protein phosphorylation by inhibiting the demethylation reactions of PP2A. High folic acid concentrations (20 and 40 μmol/L) increased SAM:SAH ratios and cell viability.. Therefore, we can speculate that folate deficiency may be a cause of PP2A deregulation, which can in turn lead to expression of the abnormal hyperphosphorylated form of tau.

Topics: Alzheimer Disease; Cell Line, Tumor; Cell Survival; Folic Acid; Folic Acid Deficiency; Humans; Methylation; Neuroblastoma; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; tau Proteins

Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.

Topics: Cell Line, Tumor; Dose-Response Relationship, Drug; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Molecular Chaperones; Neuroblastoma; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Recombinant Fusion Proteins; tat Gene Products, Human Immunodeficiency Virus; tau Proteins

Okadaic acid (OA) and dinophysistoxins (DTXs) are a group of marine toxins that cause diarrheic shellfish poisoning (DSP) in humans and animals. These compounds are produced by dinoflagellates of the Prorocentrum and Dinophysis genera and can accumulate in filter-feeding bivalves, posing a serious health risk for shellfish consumers. The enteric nervous system (ENS) plays a crucial role in the regulation of the gastrointestinal tract. In addition, neuropeptides produced by ENS affects the epithelial barrier functions. In the present work we used a two-compartment human coculture model containing the SH-SY5Y neuroblastoma cell line and polarized colonic epithelial monolayers (Caco-2) to study the OA intestinal permeability. First, we have determined OA cytotoxicity and we have found that OA reduces the viability of SH-SY5Y in a dose-dependent way, even though DTX1 is 4 to 5 times more potent than OA. Besides DTX1 is 15 to 18 orders of magnitude more potent than OA in decreasing transepithelial electrical resistance (TEER) of caco-2 cells without inducing cytotoxicity. Permeability assays indicate that OA cross the monolayer and modulates the neuropeptide Y (NPY) secretion by neuroblastoma cells. This NPY also affects the permeability of OA. This offers a novel approach to establish the influence of OA neuronal action on their diarrheic effects through a cross talk between ENS and intestine via OA induced NPY secretion. Therefore, the OA mechanisms of toxicity that were long attributed only to the inhibition of protein phosphatases, would require a reevaluation.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Survival; Diarrhea; Electric Impedance; Humans; Neuroblastoma; Neurons; Neuropeptide Y; Okadaic Acid; Pyrans

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100-500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Topics: Actin Cytoskeleton; Apoptosis; Cell Adhesion; Cell Line; Cytoskeletal Proteins; Humans; Mass Spectrometry; Membrane Microdomains; Neuroblastoma; Okadaic Acid; Phosphorylation; Signal Transduction

We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer's disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coleoptera; Cyclin-Dependent Kinase Inhibitor p27; Half-Life; Humans; Insect Proteins; Mice; Molecular Sequence Data; Neural Stem Cells; Neuroblastoma; Neurons; Neuroprotective Agents; Okadaic Acid; Oxidopamine; Peptides; Proteolysis

Tau hyperphosphorylation has been implicated in the pathogenesis of a variety of forms of human epilepsy. Here we investigated whether treatment with sodium selenate, a drug which reduces pathological hyperphosphorylated tau by enhancement of PP2A activity, would inhibit seizures in rodent models. In vitro, sodium selenate reduced tau phosphorylation in human neuroblastoma cells and reversed the increase in tau phosphorylation induced by the PP2A inhibitor, okadaic acid. Sodium selenate treatment was then tested against three different rodent seizure models. Firstly the propensity of 6-Hz electrical corneal stimulation to induce seizures in adult mice was assessed following acute treatment with different doses of sodium selenate. Secondly, the number of seizures induced by pentylenetetrazole (PTZ) was quantified in rats following chronic sodium selenate treatment via drinking water. Finally, amygdala kindled rats were chronically treated with sodium selenate in drinking water and the length and the severity of the seizures evoked by stimulation of the amygdala recorded. The results demonstrated a dose-dependent protection of sodium selenate against 6-Hz stimulation induced seizures, and significant reduction in the total number of seizures following PTZ injection. Amygdala kindled rats chronically treated with sodium selenate had significantly shorter seizure duration compared controls, with more pronounced effects observed as the duration of treatment increased. The results of this study indicate that targeting hyperphosphorylated tau by treatment with sodium selenate has anti-seizure effects in a broad range of rodent models, and may represent a novel approach to treatment of patients with epilepsy.

Topics: Amygdala; Analysis of Variance; Animals; Antioxidants; Cell Line, Tumor; Convulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Leucine; Male; Mutation; Neuroblastoma; Okadaic Acid; Pentylenetetrazole; Phosphorylation; Proline; Rats; Rats, Sprague-Dawley; Rats, Wistar; Seizures; Selenic Acid; Selenium Compounds; tau Proteins; Time Factors; Transfection

Alzheimer's disease is the most common form of dementia leading to the irreversible loss of neurons, and Tau hyperphosphorylation has an important role in the pathology of Alzheimer's disease. Ginkgolide A is one of the active components of Ginkgo biloba extracts which has been proven to have neuroprotective effects, but the effect of ginkgolide A on Tau hyperphosphorylation has not yet been reported. In this study, the effects of ginkgolide A on cell viability, Tau hyperphosphorylation, and the PI3K-Akt signaling pathway in N2a cell lines were explored, and methods such as the MTT assay, ELISA, and Western blots techniques were used. The results showed that ginkgolide A could increase cell viability and suppress the phosphorylation level of Tau in cell lysates, meanwhile, GSK3β was inhibited with phosphorylation at Ser9. Moreover, treatment of the cells with ginkgolide A promoted phosphorylation of PI3K and Akt, suggesting that the activation of the PI3K-Akt signaling pathway may be the mechanism for ginkgolide A to prevent the intracellular accumulation of p-Tau induced by okadaic acid and to protect the cells from Tau hyperphosphorylation-related toxicity.

Topics: Alzheimer Disease; Animals; Cell Line; Cell Survival; Ginkgo biloba; Ginkgolides; Lactones; Mice; Neuroblastoma; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphorylation; Plant Extracts; Signal Transduction; tau Proteins

One of pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs) consisting of abnormally hyperphosphorylated tau. The molecular mechanisms underlying the regulation of tau hyperphosphorylation remain largely unclear. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in the pathogenesis of AD, however, potential functions and role of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been fully explored. Here, we report that okadaic acid (OA)-induced tau phosphorylation is accompanied by PTEN induction, knockdown of PTEN reduces the tau hyperphosphorylation by OA in SH-SY5Y cells and increases cell proliferation and survival. The effect of PTEN suppression on tau dephosphorylation appeared to be mediated by inhibition of glycogen synthase kinase 3 while enhancing the Akt activity. Reduction of tau phosphorylation was also observed in the OA-induced parental SH-SY5Y cells co-treated with bisperoxovanadate (bpv), a potent PTEN inhibitor. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis and give some insight into the mechanisms through which suppression of PTEN expression may contribute towards the amelioration of tauopathy, implying that pharmacological intervention of PTEN may be a new therapeutic approach for the treatment of AD.

Topics: Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Humans; Membrane Proteins; Neuroblastoma; Okadaic Acid; Phosphorylation; PTEN Phosphohydrolase; RNA, Small Interfering; Signal Transduction; tau Proteins; Time Factors; Transfection

Using a neuronal model of serum starved SK-N-SH neuroblastoma cells, we showed previously that the phosphorylation of Akt and the mTOR substrates S6K and S6 through the vascular endothelial growth factor receptor VEGFR2 was enhanced by treatments with the phosphatase PP2A inhibitor okadaic acid (OA). These findings suggested that PP2A inhibition uncouples the regulation of Akt signaling by mTOR and affects cell survival. We therefore examined the effects of mTOR inhibition on Akt phosphorylation at sites threonine 308 (T308) and serine 473 (S473) and survival in OA treated cells. OA induced a loss in cell viability, the accumulation of hyperactivated Akt as monomeric and ubiquitinated forms and an increase in the total levels of ubiquitinated proteins. These events were exacerbated by treatments with an allosteric (rapamycin) but not an active-site inhibitor (PP242) of mTOR. Notably, rapamycin augmented the OA-induced hyperphosphorylation of Akt by suppressing a negative feedback loop of Akt activation through VEGFR2 and its downstream target phosphatidylinositol 3-kinase (PI3K). Treatments with the antioxidant N-acetlycysteine but not the pan caspase inhibitor Z-VAD-FMK promoted survival. Unlike reports that rapamycin promotes survival through increased Akt activation, these findings show that rapamycin-induced hyperphosphorylation of Akt fails to rescue our neuronal model from an oxidative stress-induced and caspase-independent cell death mediated by PP2A inhibition. Moreover, the exacerbation of OA-induced events by rapamycin suggests that mTOR and PP2A work in concert to regulate cell survival, activated Akt and the levels of ubiquitinated proteins.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; Immunoblotting; Immunoprecipitation; Neuroblastoma; Okadaic Acid; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor Receptor-2

Muscarinic acetylcholine receptors (mAchRs) are guanosine nucleotide-binding protein (G protein) coupled receptors that crosstalk with receptor tyrosine kinases (RTKs) to signal mitogenic pathways. In particular, mAchRs are known to couple with RTKs for several growth factors to activate the mammalian target of rapamycin (mTOR)/Akt pathway, a regulator of protein synthesis. The RTK for the vascular endothelial growth factor (VEGF), VEGFR2, can signal protein synthesis but whether it cooperates with mAchRs to mediate mTOR activation has not been demonstrated. Using serum starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2 dependent manner. Treatments with carbachol increased VEGFR2 phosphorylation, suggesting that mAchRs stimulate VEGFR2 transactivation to enhance mTOR signaling. Inhibitor studies revealed that phosphatidylinositol 3 kinase resides upstream from S6K, S6 and Akt phosphorylation while protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, okadaic acid elicited a far greater increase in phosphorylation, implicating phosphatase 2A as a critical determinant of their function. Finally, pilocarpine but not carbachol induced a time and dose dependent cell death that was associated with caspase activation and oxidative stress but independent of S6K and S6 activation through VEGFR2. Accordingly, our findings suggest that mAchRs crosstalk with VEGFR2 to enhance mTOR activity but signal divergent effects on survival through alternate mechanisms.

Topics: Apoptosis; Carbachol; Cell Line, Tumor; Culture Media, Serum-Free; Humans; Neuroblastoma; Neurons; Okadaic Acid; Phosphatidylinositol 3-Kinase; Phosphorylation; Pilocarpine; Protein Kinase C; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Receptors, Muscarinic; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases; Vanadates; Vascular Endothelial Growth Factor Receptor-2

Okadaic acid (OA) is a marine toxin produced by dinoflagellate species which is frequently accumulated in molluscs usual in the human diet. The exact action mechanism of OA has not been described yet and the results of most reported studies are often conflicting. The aim of this work was to evaluate the OA effects on morphology, cell cycle and apoptosis induction by means of light microscopy and flow cytometry, in three different types of human cells (leukocytes, HepG2 cells and SHSY5Y cells). Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction. OA induced morphological changes in all the cell types studied, and cell cycle disruption only in leukocytes and neuronal cells. SHSY5Y cells were the most sensitive to OA assault. Results obtained in the presence and absence of metabolic activation were similar, suggesting that OA acts both directly and indirectly. Furthermore, OA was found to increase the subG(1) region in the flow cytometry cell cycle analysis, suggesting induction of apoptosis. These results were confirmed by the employment of specific methodologies for studying apoptosis such as caspase 3 activation and annexin V staining. Increases in the apoptosis rate were obtained in all the cells treated in the absence of S9 fraction, accompanied by increases in caspase 3 activation, suggesting that apoptosis induced by OA is a caspase 3-dependent process. Nevertheless, in the presence of S9 fraction no apoptosis was detected, indicating a metabolic detoxifying activity, although necrosis was observed in neuroblastoma cells.

Topics: Apoptosis; Cell Cycle; Harmful Algal Bloom; Hep G2 Cells; Humans; Leukocytes; Marine Toxins; Neuroblastoma; Okadaic Acid; Water Pollutants, Chemical

A good model of neuronal death that reproduces the characteristic tau (τ) hyperphosphorylation of Alzheimeŕs disease is the use of okadaic acid (OA). The aim of this study was to determine the contribution of α7 and β2* nicotinic acetylcholine receptor (nAChR) subtypes to neuroprotection against OA in the SH-SY5Y cell line by using the selective α7 and β2* nAChR agonists PNU 282987 and 5-Iodo-A85380, respectively. The results of this study show that both α7 and β2* nAChR can afford neuroprotection against OA-induced neurotoxicity. Protection mediated by α7 nAChRs was independent of Ca(2+) and involved the intracellular signaling pathway Janus Kinase-2/Phosphatidylinositol-3-kinase/Akt. When Ca(2+) entry was promoted through the α7 nAChR by using the α7-selective positive allosteric modulator PNU 120596, protection was lost. By contrast, protection mediated by β2* nAChRs was Ca(2+) dependent and implicated the signaling pathways PI3K/Akt and extracellular regulated kinase 1/2. Both α7 and β2* nAChR activation converged on downregulation of GSK-3β and reduction of τ phosphorylation in cells undergoing cell death induced by OA. Therefore, targeting nAChR could offer a strategy for reducing neurodegeneration secondary to hyperphosphorylation of protein τ.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Azetidines; Benzamides; Bridged Bicyclo Compounds; Calcium; Cell Line, Tumor; Humans; Ionophores; Isoxazoles; Janus Kinase 2; Neuroblastoma; Neurons; Nicotinic Agonists; Okadaic Acid; Phenylurea Compounds; Pyridines; Receptors, Nicotinic; Signal Transduction

To investigate the effects of insulin-like growth factor-1 (IGF-1) on cell injuries and tau hyperphosphorylation induced by okadaic acid (OA).. The experimental groups were designed as follows: (1) SH-SY5Y culture (control group); (2) SH-SY5Y exposed to 40 nmol/L OA for 24 hours (OA group); (3) SH-SY5Y exposed to OA for 24 hours in the presence of 2 hour pretreatment with 100, 200 and 400 ng/ml IGF-1 (IGF-1 pretreatment groups). The changes of cell morphology were observed by inverted microscope. The viability of cells was detected by MTT. The injuries of cells were examined by Hoechst 33258 staining and the activity of caspase-3. Western-blot was applied to determine the expression of phosphorylation of tau protein.. In IGF-1 pretreatment group, the cell morphology was improved, the viability of cells was increased, and caspase-3 activation and hyperphosphorylation of tau (Ser396) were reduced.. IGF-1 can protect the SH-SY5Y cells from cell injuries induced by OA by inhibiting tau hyperphosphorylation.

Topics: Cell Line, Tumor; Humans; Insulin-Like Growth Factor I; Neuroblastoma; Neuroprotective Agents; Okadaic Acid; Phosphorylation; tau Proteins

1-methyl-4-phenylpyridinium ion (MPP(+)) has been shown to selectively inhibit mitochondrial function and induce a parkinsonism-like syndrome. MPP(+) stimulates the production of reactive oxygen species (ROS) and induces cell death in vitro. In this study, we investigated the protective effects of okadaic acid on MPP(+)-induced cell death in SH-SY5Y neuroblastoma cells. We found that MPP(+)-induced apoptosis and -ROS generation were blocked by okadaic acid. MPP(+)-mediated activation of AKT was also inhibited by okadaic acid. Taken together, these results demonstrate that okadaic acid protects against MPP(+)-induced apoptosis by blocking ROS stimulation and ROS-mediated signaling pathways in SH-SY5Y cells. These data indicated that okadaic acid could provide a therapeutic strategy for the treatment of neurodegenerative diseases including Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; Apoptosis; Cell Line, Tumor; Cytoprotection; Enzyme Inhibitors; Herbicides; Humans; Nerve Degeneration; Neuroblastoma; Neurons; Neuroprotective Agents; Okadaic Acid; Oxidative Stress; Parkinson Disease; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.

Topics: Analysis of Variance; Carbazoles; Cell Line, Tumor; Electric Impedance; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Humans; Laminin; Microchip Analytical Procedures; Microelectrodes; Neuroblastoma; Okadaic Acid; Phosphorylation; Staurosporine; tau Proteins

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer's disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. Here, we show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3alpha and 3beta phosphorylate purified pig brain CRMP-2 and significantly alter its mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knockdown of both GSK-3alpha and 3beta, but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3alpha-mediated phosphorylation of CRMP-2 in vitro. Collectively, our results demonstrate for the first time that OA can induce phosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3alpha and 3beta and Ser-522 kinase(s) are involved in this process.

Topics: Alzheimer Disease; Animals; Brain; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Intercellular Signaling Peptides and Proteins; Isoenzymes; Mutation; Nerve Tissue Proteins; Neuroblastoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Swine; Tissue Extracts

Several recent studies have indicated that increased levels of homocysteine (HC), including that resulting from deficiency in folate, increases tau phosphorylation. Some studies indicate that this is accomplished via HC-dependent activation of NMDA channels and resultant activation of calcium-dependent kinase pathways, while others suggest that the increase in tau phosphorylation is derived via HC-dependent inhibition of methylation of phosphatases and resultant inhibition of phosphatase activity. We demonstrate herein in SH-SY-5Y human neuroblastoma that both of these phenomena contribute to the increase in phospho-tau immunoreactivity following folate deprivation, and that supplementation with S-adenosyl methionine (SAM) prevents both the increase in kinase activity and the decrease in phosphatase activity. These findings demonstrate that the divergent neuropathological consequences of folate deprivation includes multiple pathways that converge upon tau phosphorylation, and further support the notion that dietary supplementation with SAM may reduce or delay neurodegeneration.

Topics: Calcium; Cell Line, Tumor; Drug Interactions; Enzyme Inhibitors; Folic Acid Deficiency; Homocysteine; Humans; N-Methylaspartate; Neuroblastoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Phosphorylation; S-Adenosylmethionine; tau Proteins

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B''-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.

Topics: Acetylation; Animals; Cell Line; Enzyme Inhibitors; Gene Expression Regulation; Mice; Microtubules; Mutation; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; RNA Interference; Transfection; Tyrosine

Neuronal apoptosis is involved in neurodegenerative diseases such as Alzheimer's disease and Parkinson.s disease. An efficient means of preventing it remains to be found. Some n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA, 22 : 6n-3) and eicosapentaenoic acid (EPA, 20 : 5n-3) have been reported to be protective against the neuronal apoptosis and neuronal degeneration seen after spinal cord injury (SCI) [1]. However, it is unclear which kinds of PUFAs have the most potent ability to inhibit neuronal apoptosis and whether the simultaneous treatment of PUFAs inhibits the apoptosis. In the present study, we compared the abilities of various n-3- and n-6- PUFAs to inhibit the apoptosis induced after the administration of different apoptotic inducers, etoposide, okadaic acid, and AraC, in mouse neuroblastoma cells (Neuro2a). Preincubation with DHA (22 : 6n-3), eicosapentaenoic acid (EPA, 20 : 5n-3), alpha-linolenic acid (alpha-LNA, 18 : 3n-3), linoleic acid (LA, 18 : 2n-6), arachidonic acid (AA, 20 : 4n-3), and gamma-linolenic acid (gamma-LNA, 18 : 3n-6) significantly inhibited caspase-3 activity and LDH leakage but simultaneous treatment with the PUFAs had no effect on the apoptosis of Neuro2a cells. There were no significant differences of the anti-apoptotic eff ect among the PUFAs. These results suggest that PUFAs may not be effective for inhibiting neuronal cell death after acute and chronic neurodegenerative disorders. However, dietary supplementation with PUFAs may be beneficial as a potential means to delay the onset of the diseases and/or their rate of progression.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cytarabine; Dose-Response Relationship, Drug; Etoposide; Fatty Acids, Unsaturated; Mice; Neuroblastoma; Neurons; Okadaic Acid

This study is to investigate the protective effect of (-) clausenamide against the neurotoxicity of okadaic acid in SH-SY5Y cell line, and injection beta-amyloid peptide25-35 (Abeta25-35) to the cerebral ventricle in ovariectomy (OVX) rats. MTT assay, LDH assay, and Hoechst 33258 staining were used to detect the effect of (-) clausenamide on the toxicity of okadaic acid in SH-SY5Y cell line. The animal model was induced by ovariectomized and injection of Abeta25-35 in the cerebroventricle of rats. The effect of (-) clausenamide on learning and memory deficiency was observed by step-through test. Electron microscope, Nissl body staining, and HE staining were used to examine the morphological changes in hippocampus and cerebral cortex neurons. Pretreatment of (-) clausenamide and LiCl decreased the rate of cell death from MTT, LDH release, and apoptosis from Hoechst 33258 staining in SH-SY5Y cell line. The step-through tests showed (-) clausenamide could improve the ability of learning and memory. The Nissl body staining and HE staining experiments also showed the neuroprotective effects of (-) clausenamide on the neurons of hippocampus and cerebral cortex. (-) Clausenamide has the protective effects against the neurotoxicity induced by okadaic acid and Abeta25-35.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Cerebral Cortex; Clausena; Drugs, Chinese Herbal; Female; Hippocampus; Humans; L-Lactate Dehydrogenase; Lactams; Learning; Lignans; Memory Disorders; Neuroblastoma; Neurons; Neuroprotective Agents; Okadaic Acid; Ovariectomy; Peptide Fragments; Plants, Medicinal; Rats; Rats, Sprague-Dawley

The effect of carvedilol on oxidative and cell damage induced by okadaic acid in N1E-115 cells were studied. The effects of okadaic acid were evaluated as changes in: the quantity of lipid peroxidation products, protein carbonyl groups, reduced glutathione content (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase and total lactate dehydrogenase (cell LDH). Additionally, a dose of carvedilol (10(-5)M) was added 2h before incubation with okadaic acid (50 nM) and was present until the end of the experiment (2h later added okadaic acid). Our results reveal that okadaic acid induces oxidative stress and an increase of cell LDH in N1E-115 cells, whereas carvedilol prevented the changes prompted by okadaic acid. In conclusion, the data show the protective effect of carvedilol, as well as its ability to modify cell response to okadaic acid, involving like cytoprotective mechanism its antioxidative properties.

Topics: Animals; Carbazoles; Carvedilol; Catalase; Cell Line, Tumor; Drug Interactions; Glutathione; Glutathione Peroxidase; L-Lactate Dehydrogenase; Lipid Peroxidation; Melatonin; Mice; Neuroblastoma; Okadaic Acid; Oxidation-Reduction; Oxidative Stress; Propanolamines; Proteins; Superoxide Dismutase

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.

Topics: Casein Kinase I; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Kidney; Neuroblastoma; Okadaic Acid; Phosphorylation; Protein Folding; Protein Kinase C; Protein Structure, Tertiary; Ubiquitin-Protein Ligases

Insulin receptor substrate (IRS) proteins are major docking molecules for the type I insulin like growth factor (IGF) receptor (IGF-IR) and mediate their effects on downstream signaling molecules. In this report, we investigated IRS-1 regulation during apoptosis in human neuroblastoma SH-EP cells. Treatment of SH-EP cells with mannitol or okadaic acid (OA) induces apoptosis with the typical characteristics of anoikis. Mannitol treatment results in IRS-1 degradation with concomitant appearance of smaller fragments, likely representing caspase cleavage products. In contrast OA-induced IRS-1 degradation is accompanied by a mobility shift in IRS-1, suggesting IRS-1 serine/threonine phosphorylation. Mannitol-induced, but not OA-induced, degradation is blocked by IGF-I. Pretreatment of the cells with caspase or proteasome inhibitors also partially blocks mannitol-induced IRS-1 degradation. These results suggest two independent pathways are involved in IRS-1 degradation; one pathway is dependent on caspase activation and is blocked by IGF-I, while a second pathway is caspase-independent and IGF-I-insensitive.

Topics: Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Mannitol; Neuroblastoma; Okadaic Acid; Phosphoproteins; Protease Inhibitors; Proteasome Inhibitors; Signal Transduction

Alzheimer's disease (AD) is three times more prevalent in women than men, and epidemiological studies have shown that estrogen replacement in aging women forestalls the onset of AD. Hyperphosphorylation of the tau protein that forms the neurofibrillary tangles found in AD brains might be responsible for the breakdown of microtubules in affected neurons. The mechanisms by which tau protein is phosphorylated in the AD brain are not fully understood. Using a human neuroblastoma cell line (SH-SY5Y) and primary cultures of newborn male or female rat cerebral cortical neurons, we investigated the effect of 17beta-estradiol on tau protein expression and phosphorylation. We found that estradiol increased total tau and induced dephosphorylation at the proline-directed site of the molecule. Further, estradiol prevented okadaic acid-induced hyperphosphorylation of tau in both proline- and non-proline-directed sites, and antiestrogens blocked this effect. To our knowledge, this is the first report of an effect of estradiol on naturally occurring and induced tau phosphorylation. This assumes special significance because the estrogen action was found to be sexually dimorphic in rat cortical neurons and differentiation-sensitive in human neuroblastoma cells.

Topics: Alzheimer Disease; Animals; Animals, Newborn; Binding Sites; Cell Line, Tumor; Cells, Cultured; Cerebral Cortex; Estradiol; Estrogen Antagonists; Female; Humans; Male; Neuroblastoma; Neurons; Okadaic Acid; Phosphorylation; Proline; Rats; Tamoxifen; tau Proteins

Donepezil, rivastigmine, and galantamine are three drugs with acetylcholinesterase (AChE)-inhibiting activity that are currently being used to treat patients suffering from Alzheimer's disease. We have studied the neuroprotective effects of these drugs, in comparison with nicotine, on cell death caused by beta-amyloid (Abeta) and okadaic acid, two models that are relevant to Alzheimer's pathology, in the human neuroblastoma cell line SH-SY5Y. Galantamine and donepezil showed a U-shaped neuroprotective curve against okadaic acid toxicity; maximum protection was achieved at 0.3 microM galantamine and at 1 microM donepezil; at higher concentrations, protection was diminished. Rivastigmine showed a concentration-dependent effect; maximum protection was achieved at 3 microM. When apoptosis was induced by Abeta25-35, galantamine, donepezil, and rivastigmine showed maximum protection at the same concentrations: 0.3, 1, and 3 microM, respectively. Nicotine also afforded protection against Abeta- and okadaic acid-induced toxicity. The neuroprotective effects of galantamine, donepezil, and nicotine were reversed by the alpha7 nicotinic antagonist methyllycaconitine but not by the alpha4beta2 nicotinic antagonist dihydro-beta-erythroidine. The phosphoinositide 3-kinase (PI3K)-Akt blocker 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) reversed the protective effects of galantamine, donepezil, and nicotine but not that of rivastigmine. In contrast, the bcl-2 antagonist ethyl[2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) reversed the protective effects of the three AChE inhibitors and that of nicotine. Our results show that galantamine, donepezil, and rivastigmine afford neuroprotection through a mechanism that is likely unrelated to AChE inhibition. Such neuroprotection seemed to be linked to alpha7 nicotinic receptors and the PI3K-Akt pathway in the case of galantamine and donepezil but not for rivastigmine.

Topics: Aconitine; Amyloid beta-Peptides; Apoptosis; Benzopyrans; Cell Culture Techniques; Cell Line, Tumor; Cholinesterase Inhibitors; Chromones; Donepezil; Dose-Response Relationship, Drug; Enzyme Inhibitors; Galantamine; Humans; Indans; L-Lactate Dehydrogenase; Morpholines; Neuroblastoma; Neuroprotective Agents; Nicotine; Nitriles; Okadaic Acid; Phenylcarbamates; Phosphatidylinositol 3-Kinases; Piperidines; Proto-Oncogene Proteins c-bcl-2; Receptors, Nicotinic; Rivastigmine

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.

Topics: Actins; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Cytoskeleton; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Humans; Membrane Potentials; Mitochondria; Neuroblastoma; Okadaic Acid; Pharmaceutical Preparations; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

To explore the protective effects of melatonin (Mel) on the abnormal phosphorylation of neuronal cytoskeletal proteins.. We generated a neuroblastoma (SH-SY5Y) cell system in which cytoskeletal proteins are abnormally phosphorylated resulting in microtubule disruption due to the marked inhibition of protein phosphatase activities by okadaic acid (OA).. OA-induced declines in cell viability and mitochondrial metabolic activity were remarkably prevented by Mel. In addition, the hyperphosphorylation/accumulation of neurofilament-(NF-) H/M subunits and the disruption of microtubules, induced by OA, were significantly inhibited by Mel.. Our results suggest multiple protective functions of Mel against a series of pathological lesions known to culminate in AD, including abnormal phosphorylation of cytoskeletal proteins, microtubule disassembly and mitochondrion-initiated cell toxicity.

Topics: Alzheimer Disease; Cell Survival; Humans; Melatonin; Microtubules; Mitochondria; Neuroblastoma; Neurofilament Proteins; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Tumor Cells, Cultured

Progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, both in neural aging and in neurodegenerative diseases. Cytoskeleton in neurodegenerative diseases is characterized by hyperphosphorylated tau assembled in neurofibrillary tangles. Tau protein promotes microtubule enlargement and its hyperphosphorylation inhibits tubulin assembly. Okadaic acid (OA) causes oxidative stress, tau hyperphosphorylation, and altered cytoskeletal organization similar to those observed in neurons of patients with dementia. Since melatonin acts by both enlarging microtubules and as a free-radical scavenger, in this work we studied the effects of melatonin on altered cytoskeletal organization induced by OA in N1E-115 neuroblastoma cells. Optic microscopy, morphometric analysis, and tubulin immunofluorescence staining of neuroblastoma cells incubated with 50 nM OA showed an intact microtubule network following the neurite profile similar to that observed in the vehicle-incubated cells when melatonin was added to the incubation media 2 h before OA. The melatonin effects on altered cytoskeletal organization induced by OA were dose-dependent and were not abolished by luzindole, the mt(1) melatonin antagonist receptor. Also, increased lipid peroxidation and augmented apoptosis in N1E-115 cells incubated with 50 nM OA were prevented by melatonin. The results support the hypothesis that melatonin can be useful in the treatment of neurodegenerative diseases.

Topics: Animals; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Melatonin; Mice; Microtubules; Neuroblastoma; Okadaic Acid; Oxidative Stress; Pertussis Toxin; Phosphoric Monoester Hydrolases; Time Factors; Tumor Cells, Cultured

Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimer's disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.

Topics: Antibodies, Monoclonal; Antigens; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Densitometry; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Flavonoids; Fluorescent Antibody Technique; Humans; Immunoblotting; Intracellular Space; Neuroblastoma; Nimodipine; Nuclear Proteins; Okadaic Acid; Phosphorylation; Protein Subunits; Sirtuin 2; Sirtuins; tau Proteins; Tumor Cells, Cultured

To explore if protective effect of melatonin on oxidative stress induced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, is mediated by membrane receptors subtype mt1, we used an in vitro model with N1E-115 neuroblastoma cells. We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induces a reduction in the activity of antioxidative enzymes, and an increase of lipid peroxidation products, while melatonin prevents the effect of okadaic acid. On the other hand, the presence of luzindole, 20 min before adding melatonin, did not cause changes on the effect of the melatonin on oxidative stress. These results seem to indicate that protective effect of melatonin is not mediated by mt1 receptors.

Topics: Animals; Cell Line, Tumor; Enzyme Inhibitors; Lipid Peroxidation; Melatonin; Mice; Neuroblastoma; Okadaic Acid; Oxidative Stress; Receptor, Melatonin, MT1; Tryptamines

We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induced a reduction in cellular glutathione transferase, glutathione reductase and catalase activity. Likewise, this acid prompted an increase in lipid peroxidation. Treatment of cells with 10(-5) M melatonin or 0.5 microg/ml vitamin C prevented the effects of okadaic acid. These results indicate that okadaic acid induces an oxidative stress imbalance, while melatonin and vitamin C prevent the oxidative stress induced by okadaic acid. Likewise, these data indicate the great importance of oxidative stress in both this experimental model and in the development and course of neurodegenerative disease, especially Alzheimer's disease. They show that melatonin is much more efficient than vitamin C in reducing the extent of oxidative stress. This phenomenon was demonstrated by the smaller dose of melatonin needed to obtain effects similar to those obtained with vitamin C on lipid peroxidation and by the protective effect of melatonin on antioxidant enzyme activity.

Topics: Alzheimer Disease; Animals; Ascorbic Acid; Catalase; Glutathione Transferase; Lipid Peroxidation; Melatonin; Mice; Neuroblastoma; Okadaic Acid; Oxidative Stress; Tumor Cells, Cultured

Apoptotic changes induced by okadaic acid and yessotoxin in BE(2)-M17 neuroblastoma cells have been evaluated and quantified by combining classical methods and fast and sensitive fluorimetric microplate assays. The phosphatase inhibitor okadaic acid induced rapid time- and dose-dependent apoptotic changes in this cell line, which were evident after 1h at concentrations equal or higher than 500 nM. Decreased mitochondrial membrane potential by okadaic acid (IC(50)=350 nM at 1h) was followed by cell detachment (IC(50)=400 nM at 1h), changes in total nucleic acids content (50% of controls after 1h with 1000 nM okadaic acid), caspase-3 activation (3- to 4-fold increase at 6h) and increased Annexin-V binding (1.5-fold at 6h). Yessotoxin induced similar changes in BE(2)-M17 cells, although significant differences were found in the time-course and degree of apoptotic events induced by this phycotoxin, indicating a lower potency for yessotoxin when compared with okadaic acid. This is the first report on apoptogenic activity of yessotoxin.

Topics: Annexin A5; Apoptosis; Caspase 3; Caspases; Cell Adhesion; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Intracellular Membranes; Membrane Potentials; Mitochondria; Mollusk Venoms; Neuroblastoma; Okadaic Acid; Oxocins; RNA, Neoplasm; 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

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).

Topics: Cell Death; Crk-Associated Substrate Protein; Cyclosporine; Enzyme Inhibitors; Humans; Molsidomine; Neuroblastoma; Nitric Oxide Donors; Okadaic Acid; Peroxynitrous Acid; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Protein Tyrosine Phosphatases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-crk; Retinoblastoma-Like Protein p130; Signal Transduction; Tumor Cells, Cultured; Vanadates

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Serl99/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.

Topics: Animals; Calcineurin; Cell Line; Enzyme Inhibitors; Humans; Neuroblastoma; Okadaic Acid; PC12 Cells; Pheochromocytoma; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Subunits; Rats; Recombinant Proteins; tau Proteins; Transfection; Tumor Cells, Cultured

To study the regulatory effect and possible mechanism of Tiaoxin Recipe (TXR) on animal's Alzheimer disease related tau protein phosphorylation.. NG108 cell model was treated with Okadaic acid and related parameters were determined using MTT staining, immunoblot, coimmunoprecipitation assay, etc.. Shown by MTT staining, NG108 cell activity decreased significantly after treated with Okadaic acid for 12 hrs, which could be ameliorated by TXR rat serum. Revealed by immunoblot method, the Okadaic acid induced elevation of phosphorylated tau protein could partly be reversed after co-treated with TXR rat serum. TXR extract could inhibit the binding of tau protein with presenilin-1, which may regulate the tau protein phosphorylation, and could be observed by coimmunoprocipitation.. TXR could inhibit tau protein hyperphosphorylation, which might partially be due to the TXR caused binding of presenilin-1 with tau protein.

Topics: Alzheimer Disease; Animals; Drugs, Chinese Herbal; Glioma; Male; Membrane Proteins; Mice; Neuroblastoma; Okadaic Acid; Phosphorylation; Phytotherapy; Plasma; Presenilin-1; Rats; tau Proteins; Tumor Cells, Cultured

Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Choline O-Acetyltransferase; Enzyme Induction; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Mice; Neoplasm Proteins; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Neuronal apoptosis is involved in several pathological conditions of the brain. Using cDNA arrays, we observed upregulation of ubiquitin-binding protein p62 expression during serum withdrawal-induced apoptosis in Neuro-2a cells. We demonstrate here that the expression levels of p62 mRNA and protein were increased in Neuro-2a cells and cultured rat hippocampal neurons by different types of proapoptotic treatments, including serum deprivation, okadaic acid, etoposide, and trichostatin A. Ubiquitin-binding protein p62 is a widely expressed cytoplasmic protein of unclear function. The ability of p62 to bind noncovalently to ubiquitin and to several signalling proteins suggests that p62 may play a regulatory role connected to the ubiquitin system. Accordingly, we show that proteasomal inhibitors MG-132, lactacystin, and PSI caused a prominent upregulation of p62 mRNA and protein expression, with a concomitant increase in ubiquitinated proteins. To conclude, p62 upregulation appears to be a common event in neuronal apoptosis. Results also suggest that the induction of p62 expression by proteasomal inhibitors may be a response to elevated levels of ubiquitinated proteins, possibly constituting a protective mechanism.

Topics: Animals; Apoptosis; Carrier Proteins; Cells, Cultured; Culture Media, Serum-Free; Cysteine Endopeptidases; Etoposide; Gene Expression Regulation; Hippocampus; Hydroxamic Acids; Immediate-Early Proteins; Mice; Multienzyme Complexes; Neuroblastoma; Neurons; Okadaic Acid; Proteasome Endopeptidase Complex; Rats; RNA, Messenger; Transcription Factor TFIIH; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

1. The regulation of Maxi Cl(-) channels by 17beta-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl(-) channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. 2. Whole-cell Maxi Cl(-) currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. 3. Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl(-) channels. The inhibitory effect of 17beta-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. 4. Current activation was unaffected by the removal of intracellular Ca(2+) and Mg(2+), but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl(-) channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. 5. Although the role of these Maxi Cl(-) channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.

Topics: Animals; Carcinogens; Chloride Channels; Chlorides; Cyclic AMP; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Ion Channel Gating; Mice; Neuroblastoma; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Selective Estrogen Receptor Modulators; Signal Transduction; Staurosporine; Stilbenes; Tamoxifen; Toremifene; Tumor Cells, Cultured

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation-dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF-H and NF-M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF-H and NF-M was 1.7- and 1.5-fold (P<0.01) as determined by monoclonal antibody SMI33, and was 1.6-fold (P<0.01) in NF-L using antibody NR4. The phosphorylation of NF-H and NF-M in AD was increased respectively at the SMI31 epitope by 1.6- and 1.9-fold (P<0.05) and at the SMI33 epitope by 2.7- and 1.3-fold (P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)-2A and -1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP-2A/PP-1 activities can lead to the hyperphosphorylation of NF-H and NF-M subunits.

Topics: Aged; Alzheimer Disease; Brain; Case-Control Studies; Enzyme Inhibitors; Humans; Huntington Disease; Middle Aged; Neuroblastoma; Neurofilament Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Subunits; Subcellular Fractions; Tumor Cells, Cultured

L-Deprenyl, an irreversible MAO-B (monoamine oxidase B, EC 1.4.3.4) inhibitor, is used for the treatment of Parkinson's disease and to delay the progression of Alzheimer's disease. L-Deprenyl also exhibits protective effects against neuronal apoptosis which are independent of its ability to inhibit MAO-B. The purpose of this study was to compare the antiapoptotic efficacy of L-deprenyl against different types of apoptotic inducers in three neuronal cell culture models. The level of apoptosis was quantified by measuring the activation of caspase-3 enzyme, which is the main apoptotic executioner in neuronal cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and LDH (lactate dehydrogenase, EC 1. 1.1.27) assays were used to demonstrate the cytotoxic response of apoptotic treatments. Our results showed that okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induced a prominent increase in caspase-3 activity both in cultured hippocampal and cerebellar granule neurons as well as in Neuro-2a neuroblastoma cells. Interestingly, L-deprenyl offered a significant protection against the apoptotic response induced by okadaic acid in all three neuronal models. The best protection appeared at the concentration level of 10(-9) M. L-Deprenyl also provided a protection against apoptosis after AraC (cytosine beta-D-arabinoside) treatment in hippocampal neurons and Neuro-2a cells and after etoposide treatment in Neuro-2a cells. However, L-deprenyl did not offer any protection against apoptosis caused by serum withdrawal or potassium deprivation. Okadaic acid treatment in vivo is known to induce an Alzheimer's type of hyperphosphorylation of tau protein, formation of beta-amyloid plaques, and a severe memory impairment. Our results show that the okadaic acid model provides a promising tool to study the molecular basis of Alzheimer's disease and to screen the neuroprotective capacity of L-deprenyl derivatives.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cerebellum; Drug Interactions; Enzyme Inhibitors; Etoposide; Hippocampus; Mice; Neuroblastoma; Neurons; Okadaic Acid; Protective Agents; Rats; Rats, Wistar; Selegiline; Tumor Cells, Cultured

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.

Topics: Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Glioma; Hybrid Cells; Molecular Sequence Data; Neuroblastoma; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Serine

Exposure of mouse NB-2a neuroblastoma cells to genotoxic (etoposide or cytosine arabinoside) or nongenotoxic challenges (serum deprivation or okadaic acid) resulted in progressive cell death with biochemical and morphological characteristics typical of apoptosis. Apoptotic cell death induced by nongenotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the formation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompanied apoptosis induced by genotoxic agents. Combination of genotoxic and nongenotoxic insults, i.e. incubation of etoposide-treated cells in the serum-free medium, resulted in an additive effect on the profile of DNA disintegration, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-deprived cells. On the other hand, incubation of serum-deprived cells in the presence of Zn2+-ions led to the abrogation of internucleosomal DNA fragmentation but accumulation of HMW-DNA fragments. Differences in the pattern of DNA fragmentation were reproducible in a cell free apoptotic system after treatment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free experiments also revealed that activities responsible for the formation of HMW- and oligonucleosomal-DNA fragments are separable in cytosolic extract prepared from the serum-deprived cells. Finally, DNA fragmentation induced by nongenotoxic apoptotic inducers was effectively prevented by cycloheximide and suramin, while both cycloheximide and suramin had only a slight inhibitory effect on DNA fragmentation induced by genotoxic agents. The results presented suggest that distinct pathways underlay disintegration of nuclear DNA during apoptosis induced by genotoxic and nongenotoxic inducers, and that the formation of HMW- and oligonucleosomal-DNA fragments proceeds via separate mechanisms in NB-2a neuroblastoma cells.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspases; Cytarabine; DNA; DNA Fragmentation; Etoposide; Mice; Neuroblastoma; Okadaic Acid; Tumor Cells, Cultured

Fetal Alz-50 clone 1 (FAC1) is a novel DNA binding protein with altered expression and subcellular localization during neuronal development and degeneration. FAC1 localizes to the cell body and neurites in undifferentiated neurons during development and in degenerating neurons during Alzheimer's disease progression. In the normal adult brain FAC1 is present predominantly in the nucleus of cortical neurons. When in the nucleus FAC1 has been shown to repress transcription by binding a specific DNA sequence. In the present study we demonstrate that the affinity of FAC1 for the identified DNA sequence is dramatically enhanced when FAC1 is phosphorylated. Phosphatase treatment of neuroblastoma nuclear extracts reduces FAC1 DNA binding affinity. Finally, inhibition of cellular serine/threonine phosphatases results in increased FAC1 DNA binding activity. These data suggest that FAC1 DNA binding activity is dependent upon and regulated by phosphorylation signals in the cell.

Topics: Acid Phosphatase; Adenosine Triphosphate; Animals; Antigens, Nuclear; Cell Nucleus; DNA; DNA-Binding Proteins; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurons; Nuclear Proteins; Okadaic Acid; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Rats; Recombinant Fusion Proteins; Response Elements; Transcription Factors; Tumor Cells, Cultured

O-Linked N-Acetylglucosamine (O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.

Topics: Acetylglucosamine; Animals; COS Cells; Galactose; HeLa Cells; Humans; Neuroblastoma; Okadaic Acid; Subcellular Fractions; Tritium; Tumor Cells, Cultured

In Alzheimer disease brain the microtubule associated protein (MAP) tau is abnormally hyperphosphorylated. The role of protein phosphatases (PP) in the regulation of phosphorylation of tau was studied in undifferentiated SY5Y cells. In cells treated with 10 nM okadaic acid (OA), a PP-2A/PP-1 inhibitor, the PP-1 and -2A activities decreased by 60% and 100% respectively and the activities of MAPKs, cdc2 kinase and cdk5, but not of GSK-3, increased. OA increased the phosphorylation of tau at Thr-231/Ser-235 and Ser-3961404, but not at Ser-262/356 or Ser-199/202. An increase in tyrosinated/detyrosinated tubulin ratio, a decrease in the microtubule binding activities of tau, MAP1b and MAP2, and cell death were observed. Treatment with 1 microm taxol partially inhibited the cell death. These data suggest (1) that OA induced hyperphosphorylation of tau is probably the result of activated MAPK and cdks in addition to decreased PP-2A and PP-1 activities and (2) that in SY5Y cells the OA induced cell death is associated with a decrease in stable microtubules.

Topics: Cell Survival; Cyclosporine; Enzyme Inhibitors; Humans; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Okadaic Acid; Paclitaxel; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Proline; Protein Binding; tau Proteins; Tubulin; Tumor Cells, Cultured

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.

Topics: Animals; Bradykinin; Calcium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glioma; Hybrid Cells; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxazoles; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Thapsigargin; Type C Phospholipases

Valproic acid (VPA), a simple branched fatty acid anticonvulsant, has been demonstrated to have clinical efficacy in the treatment of manic-depressive illness (Bowden et al., 1994), but the mechanism(s) by which VPA produces its therapeutic effects remain to be elucidated. VPA's clinical antimanic action require a lag period for onset and are not immediately reversed upon discontinuation of treatment, effects that suggest alterations at the genomic level; we therefore investigated the effects of VPA on the modulation of the DNA binding activity of key transcription factors. DNA binding activities of activator protein 1 (AP-1) and cAMP responsive element binding protein (CREB) were studied in acute (hours) and chronic (days) VPA-treated rat C6 glioma cells. VPA did not affect CREB DNA binding activity, but concentration- and time-dependently increased AP-1 DNA binding activity. The activity was raised at 2 hours (the shortest time examined) and remained high after 6 days (the longest time used) of continuing VPA treatment. VPA also enhanced AP-1 DNA binding activity in human neuroblastoma (SH-SY5Y) cells. Because the effects of VPA were markedly inhibited by cycloheximide, they appear to require new protein synthesis. Taken together, the data suggest that antimanic agents may affect gene expression by modulation of the activity of major transcription factors; in view of the key roles of these nuclear transcription regulatory factors in long-term neuronal plasticity and cellular responsiveness, these effects may play a major role in VPA's therapeutic efficacy and are worthy of further study.

Topics: Activating Transcription Factor 2; Animals; Anticonvulsants; Brain; Cyclic AMP Response Element-Binding Protein; Cycloheximide; DNA, Neoplasm; Dose-Response Relationship, Drug; Glioma; Humans; Indoles; Maleimides; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Protein Synthesis Inhibitors; Rats; Time Factors; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured; Valproic Acid

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydro-derivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

Topics: Animals; Cell Differentiation; Ceramides; Cytosol; Kinetics; Mice; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Sphingosine; Tretinoin; Tumor Cells, Cultured

Here we report three experimental paradigms in which tau proteins are differentially localized and expressed in human neuroblastoma cells SH-SY5Y. We found that in undifferentiated cells, tau proteins were predominantly localized in the nucleus. Western blot analysis of nuclear extracts revealed, among the others, a high molecular weight tau isoform and evaluation of tau mRNA levels showed a single tau isoform. After differentiation, tau immunoreactivity was detected only in cytosol and along neuritic processes. The high molecular weight tau isoform disappeared and an additional tau mRNA species was detected. Treatment of differentiated cells with doxorubicin or okadaic acid resulted in an increase of tau immunoreactivity and in a subsequent cell loss. Our results indicate that both subcellular localization and pattern of expression of tau proteins vary depending on the developmental and functional state of the cells, thus suggesting different roles in cell function.

Topics: Antibiotics, Antineoplastic; Cell Death; Cell Differentiation; Doxorubicin; Enzyme Inhibitors; Humans; Immunohistochemistry; Neuroblastoma; Neurons; Okadaic Acid; tau Proteins; Tumor Cells, Cultured

The abnormal cytoskeletal organization observed in Alzheimer's disease has been suggested to arise from hyperphosphorylation of tau and the resultant elimination of its ability to associate with microtubules. This possibility has been supported by a number of studies under cell-free conditions utilizing various kinases, phosphatases and their corresponding inhibitors each, and by treatment of intact cells with kinase and phosphatase activators and inhibitors. However, in studies utilizing intact cells, it remained difficult to attribute microtubule compromise specifically to tau hyperphosphorylation due to potential influence of inhibitors on tubulin and/or other microtubule-associated proteins which themselves possess assembly-regulatory phosphorylation sites. To address this difficulty, we subjected SH-SY-5Y human neuroblastoma cells to treatment with the phosphatase inhibitor okadaic acid (OA), which has been previously demonstrated to depolymerize microtubules in these cells. OA induced an increase in tau hyperphosphorylation as evidenced by an increase in Alz-50 immunoreactivity and a corresponding decrease in Tau-1 immunoreactivity. When tau-enriched fractions from OA-treated cells were incubated under microtubule assembly-promoting conditions with twice-cycled, tau-free preparations of bovine brain tubulin not exposed to OA, Alz-50-immunoreactive tau isoforms displayed a marked (49%) reduction in ability to co-assemble with bovine microtubules as compared with Tau-1- and 5E2-immunoreactive isoforms. These data indicated that hyperphosphorylated tau has a reduced capacity to associate with microtubules, and support the hypothesis that tau hyperphosphorylation may underlie microtubule breakdown in Alzheimer's disease.

Topics: Animals; Antigens; Brain; Cattle; Enzyme Inhibitors; Ethers, Cyclic; Hot Temperature; Humans; Microtubules; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; tau Proteins; Tubulin; Tumor Cells, Cultured

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Sequence; Animals; Brain Neoplasms; Calcineurin; Calmodulin-Binding Proteins; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Glioma; Half-Life; Molecular Sequence Data; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Serine-Threonine Kinases; Rats; Receptors, Serotonin; Staurosporine; Tumor Cells, Cultured

1. Activation of human D2(s) dopamine receptors with quinpirole (10 nM) inhibits omega-conotoxin GVIa-sensitive, high-threshold calcium currents when expressed in differentiated NG108-15 cells (55% inhibition at +10 mV). This inhibition was made irreversible following intracellular dialysis with the non-hydrolysable guanosine triphosphate analogue GTP-gamma-S (100 microM), and was prevented by pretreatment with pertussis toxin (1 microgram ml-1 for 24 h). 2. Stimulation of protein kinase C with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (100 microM), also attenuated the inhibition of the sustained calcium current but did not affect the receptor-mediated decrease in rate of current activation. Similarly, okadaic acid (100 nM), a protein phosphatase 1/2A inhibitor, selectively occluded the inhibition of the sustained current. 3. The depression of calcium currents by quinpirole (10 nM) was enhanced following intracellular dialysis with 100 microM cyclic adenosine monophosphate (cyclic AMP, 72.8 +/- 9.8% depression), but was not mimicked by the membrane permeant cyclic GMP analogue, Sp-8-bromoguanosine-3',5':cyclic monophosphorothioate (100 microM). 4. Inhibition of calcium currents was only partly attenuated by 100 ms depolarizing prepulses to +100 mV immediately preceding the test pulse. However, following occlusion of the sustained depression with okadaic acid (100 nM) the residual kinetic slowing was reversed in a voltage-dependent manner (P < 0.05). 5. Thus pertussis toxin-sensitive G-proteins liberated upon activation of human D2(short) dopamine receptors inhibited high-threshold calcium currents in two distinct ways. The decrease in rate of calcium current activation involved a voltage-dependent pathway, whereas the sustained inhibition of calcium current involved, in part, the voltage-resistant phosphorylation by cyclic AMP-dependent protein kinases and subsequent dephosphorylation by protein phosphatases 1/2A.

Topics: Calcium; Calcium Channel Blockers; Calcium Channels; Cyclic AMP; Diglycerides; Dopamine Agonists; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Ergolines; Ethers, Cyclic; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Neurons; Okadaic Acid; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pertussis Toxin; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Phosphatase 1; Quinpirole; Receptors, Dopamine D2; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

We investigated the intracellular events involved in the 3,3',5-triiodo-L-thyronine (T3)-induced accumulation in acetylcholinesterase (AChE) activity in neuroblastoma cells (neuro-2a) that overexpress the human thyroid receptor beta 1 (hTR beta 1). Treatment of these cells with T3 increased AChE activity and its mRNAs after a lag period of 24-48 h, and these levels increased through stabilization of the transcripts by T3. T3 had no effect on the transcriptional rate or processing of AChE transcripts. The protein kinase inhibitor H7 inhibited T3-induced accumulation in AChE activity and its mRNAs, whereas okadaic acid (a potent inhibitor of phosphatases 1 and 2A) potentiated the effect of T3. Okadaic acid and H7 have no effect on the binding of hTR beta 1 to T3 or the transcriptional rate of the AChE gene. Finally, treatment of cells with T3 stimulated cytosolic serine/threonine, but not tyrosine kinase, activities. The time course analysis reveals that the increase in serine/threonine activity precedes the effect of T3 on AChE mRNAs. These results suggest that activation of a serine/threonine protein kinase pathway might be a link between nuclear thyroid hormone receptor activation and stabilization of AChE mRNA.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholinesterase; Animals; Cell Line; Dichlororibofuranosylbenzimidazole; Enzyme Inhibitors; Ethers, Cyclic; Humans; Isoquinolines; Kinetics; Mice; Naphthalenes; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Piperazines; Protein Binding; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Receptors, Thyroid Hormone; Recombinant Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Triiodothyronine; Tumor Cells, Cultured

To investigate the regulation of posttranslational modifications of tau that might be pertinent to the production of the paired helical filament (PHF) of Alzheimer's disease, we incubated human neuroblastoma cells with the protein phosphatase inhibitor okadaic acid. This treatment results in increased immunoreactivity of tau with the monoclonal antibodies Alz-50, PHF-1, T3P, and NP8, a reduction in Tau-1 immunoreactivity, and an elevation in apparent molecular weight of tau. Moreover, our data demonstrate that accumulation of phosphates in tau leads to a decrease in the turnover rate of tau in the neuroblastoma cells. It is suggested that similar build-up of hyperphosphorylated tau in the neuronal perikarya may represent an early event in PHF formation. The present system facilitates the investigation of regulatory mechanisms governing the occurrence of PHF epitopes, their effects on neuronal cell metabolism, and possible pharmacological intervention.

Topics: Alkaline Phosphatase; Dose-Response Relationship, Drug; Epitopes; Ethers, Cyclic; Humans; Methionine; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Proteins; tau Proteins; Time Factors; Tumor Cells, Cultured

Cross-regulation from the stimulatory phospholipase C to the adenylyl cyclase pathways was explored in neuroblastoma-glioma NG-108-15 cells in culture. Activation of protein kinase C by phorbol myristic acid resulted in a markedly attenuated activation of the inhibitory adenylyl cyclase response to delta-opiate agonists and epinephrine but not to the muscarinic agonist carbachol. The ability of okadaic acid to mimic the effects of phorbol myristic acid on the inhibitory response suggested a role for protein phosphorylation. Adenylyl cyclase activity from cells in which protein kinase C had been activated demonstrated a loss in the inhibitory adenylyl cyclase response at the level of the G-protein. Activation of protein kinase C prompted a 2-4-fold increase in phosphorylation of G1 alpha 2 in cells metabolically labeled with [32P]orthophosphate. The phosphate content of Gi alpha 2 was determined to be approximately 0.5 mol/mol subunit in the unstimulated cells and approximately 1.5 mol/mol subunit for cells in which protein kinase C was activated. The effects of okadaic acid, 4-alpha-phorbol, and calphostin C on inhibition of adenylyl cyclase in cells treated with phorbol myristic acid correlate with the effects of these agents on phosphorylation of Gi alpha 2. The time courses for attenuation of inhibitory adenylyl cyclase and that for phosphorylation of Gi alpha 2 were similar in cells challenged with phorbol myristic acid. These data argue for cross-regulation from the stimulatory protein kinase C to inhibitory adenylyl cyclase pathways at the level of Gi alpha 2 via protein phosphorylation.

Topics: Adenylyl Cyclases; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Ethers, Cyclic; Glioma; GTP-Binding Proteins; Hybrid Cells; Naphthalenes; Neuroblastoma; Okadaic Acid; Phorbols; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In this study, the signal cascade transducing carbachol stimulation into c-fos expression in SH-SY5Y neuroblastoma cells was investigated. 1,2-Diacylglycerol formation and c-fos expression were mediated via stimulation of muscarinic M1 receptors and the first 5 min of receptor stimulation were critical for these events. Application of 1,2-dioctanoylglycerol induced c-fos expression and this, as well as carbachol-stimulated c-fos expression, was inhibited by protein kinase C inhibitors. Increasing the intracellular Ca2+ concentration had only small effects on c-fos expression. There was a dependency on extracellular Ca2+ for maximal c-fos expression and 1,2-diacylglycerol formation. The carbachol-stimulated c-fos expression was potentiated by application of the protein phosphatase inhibitor okadaic acid. These results demonstrate the importance of 1,2-diacylglycerol formation for muscarinic receptor-stimulated, protein kinase C-mediated c-fos expression in the SH-SY5Y cells and that this cascade is counteracted by an okadaic acid-sensitive protein phosphatase.

Topics: Carbachol; Diglycerides; Ethers, Cyclic; Gene Expression Regulation; Genes, fos; Humans; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Receptors, Muscarinic; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Recently, a mitogen activated protein kinase has been implicated in the generation of a phosphorylated paired helical filament (PHF) epitope recognized by the monoclonal antibody AT8. This epitope consists of phosphorylated serines 199 and/or 202 of the human microtubule associated protein tau. Theoretically, aside from abnormal kinase activity, inhibition of phosphatase activity could also be involved in the abnormal phosphorylation status of the microtubule associated protein tau. To investigate this, we incubated LA-N-5 neuroblastoma cells with okadaic acid, a specific inhibitor of phosphatase 2A. We found that incubating neuroblastoma cells with okadaic acid induces the abnormally phosphorylated AT8 epitope. The effect of okadaic acid is time and dose dependent and is reversible. Our findings suggest that phosphatase activity is important in the regulation of the phosphorylation state of tau. Phosphatases may act directly on tau or may influence the activity of mitogen activated protein kinase. Incubation of LA-N-5 neuroblastoma cells with okadaic acid provides a cellular model in which the generation of a well-defined PHF-tau epitope can be investigated.

Topics: Epitopes; Ethers, Cyclic; Humans; Immunoblotting; Neuroblastoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Phosphorylation; tau Proteins; Tumor Cells, Cultured

Protein phosphorylation and subsequent dephosphorylation was studied in digitonin-permeabilized neuroblastoma SH-SY5Y cells by measuring the incorporation of [32P]phosphate into myelin basic protein (MBP). 1,2-Dioctanoyl-sn-glycerol (DOG) and calcium synergistically induced phosphorylation of MBP, which was inhibited by the protein kinase C (PKC) pseudosubstrate peptide (PKC19-36). The phosphorylation increased for 10 min when a net dephosphorylation started to appear. The dephosphorylation was inhibited by okadaic acid. Regardless of calcium concentration, the presence of DOG was necessary for significant effects of okadaic acid on MBP phosphorylation. H7 and staurosporine dose-dependently inhibited the phosphorylation of MBP, induced by DOG and calcium in the presence of okadaic acid. Different PKC pseudosubstrate peptides were applied and all showed an inhibitory effect on the phosphorylation of MBP under these conditions. These results demonstrate the presence, in SH-SY5Y cells, of a protein phosphatase, possibly protein phosphatase 2A, with a high basal activity that counteracts PKC-induced phosphorylation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Amino Acid Sequence; Calcium; Cell Membrane Permeability; Diglycerides; Ethers, Cyclic; Humans; Isoenzymes; Isoquinolines; Molecular Sequence Data; Myelin Basic Protein; Neoplasm Proteins; Neuroblastoma; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Polymyxin B; Protein Kinase C; Protein Phosphatase 2; Protein Processing, Post-Translational; Signal Transduction; Staurosporine; Substrate Specificity; Tumor Cells, Cultured

In Alzheimer's disease, Tau proteins are abnormally phosphorylated. In this paper, we describe a cellular model producing such pathological Tau proteins. After differentiation by NGF and treatment with okadaic acid (an inhibitor of phosphatases 1 and 2 A), neuroblastoma SKNSH-SY 5Y cells produced Tau proteins with an increased apparent molecular weight and a more acidic isoelectric point when compared to Tau proteins from control cells. These modified tau proteins bore Alzheimer-type epitopes detectable by antibodies specific to phosphorylated Alzheimer epitopes. This model is the first step toward a pharmacological approach of neuroprotection.

Topics: Alzheimer Disease; Epitopes; Ethers, Cyclic; Humans; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; tau Proteins; Tumor Cells, Cultured

Human neuroblastoma cells, LAN, were used to study the phosphorylation and dephosphorylation of tau proteins. These cells contained mainly a form of tau comparable to fetal brain tau in molecular weight (55 kDa). Neuroblastoma tau reacted with antibodies that recognize epitopes spanning the whole tau molecule (E-1, Alz50, Tau-1, and Tau46), and antibodies (PHF-1, NP8, and T3P) that recognize hyperphosphorylated tau (PHF-tau) in Alzheimer's disease (AD) brains. Exposure of the cells to 45 degrees C heat stress resulted in dephosphorylation of the epitopes recognized by PHF-1, NP8, and T3P. Transfer of the heat-stressed cells to 37 degrees C led to rephosphorylation of the dephosphorylated epitopes. Cells that had been treated with okadaic acid (OA), regardless of whether they were subsequently subjected to heat stress or heat stress and recovery, all contained tau with a molecular weight similar to that of control cells. These tau proteins, similar to tau in control cells, also reacted with antibodies to phosphorylated epitopes. However, unlike the tau from control or heat-stressed cells, the OA-treated and heat-stressed tau had decreased reactivity with Tau-1. Alteration of Tau-1 immunoreactivity has been reported to be an early event in AD neurodegeneration. The reduction of Tau-1 immunoreactivity observed in OA-treated samples could be restored by incubation of electroblots of isolated tau with alkaline phosphatase, indicating an induction of the Tau-1 epitope phosphorylation by OA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alzheimer Disease; Cell Line; Electrophoresis, Polyacrylamide Gel; Epitopes; Ethers, Cyclic; Hot Temperature; Humans; Immunoblotting; Neuroblastoma; Okadaic Acid; Phosphorylation; tau Proteins; Tumor Cells, Cultured

We report the induction of apoptosis in a human neuroblastoma cell line SK-N-BE(2) by cisplatin or retinoic acid, and its relation to cell cycle. Apoptosis was monitored by counting apoptotic bodies and evaluating the activity of 'tissue' transglutaminase (EC 2.3.2.13), one of the genes specifically expressed in apoptotic cells. Data indicate that both agents enhance apoptosis, even though cells arrest at different cell cycle phases. In fact, retinoic acid causes accumulation in G1, whilst cisplatin induces accumulation of cells in the G2/M phase. This evidence suggests the presence of multiple start points for the apoptotic death programme within the cell cycle of human neuroblastoma cells.

Topics: Apoptosis; Caffeine; Cell Cycle; Cisplatin; Ethers, Cyclic; Humans; Interphase; Mitosis; Neuroblastoma; Okadaic Acid; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Microtubules and their associated proteins play a prominent role in many physiological and morphological aspects of brain function. Abnormal deposition of the microtubule-associated proteins (MAPs), MAP2 and tau, is a prominent aspect of Alzheimer's disease. MAP2 and tau are heat-stable phosphoproteins subject to high rates of phosphorylation/dephosphorylation. The phosphorylation state of these proteins modulates their affinity for tubulin and thereby affects the structure of the neuronal cytoskeleton. The dinoflagellate toxin okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A. In cultured rat cortical neurons and a human neuroblastoma cell line (MSN), okadaic acid induces increased phosphorylation of MAP2 and tau concomitant with early changes in the neuronal cytoskeleton and ultimately leads to cell death. These results suggest that the diminished rate of MAP2 and tau dephosphorylation affects the stability of the neuronal cytoskeleton. The effect of okadaic acid was not restricted to neurons. Astrocytes stained with antibodies to glial fibrillary acidic protein (GFAP) showed increased GFAP staining and changes in astrocyte morphology from a flat shape to a stellate appearance with long processes.

Topics: Animals; Cells, Cultured; Cerebral Cortex; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Ethers, Cyclic; Humans; Immunoblotting; Microtubule-Associated Proteins; Nerve Degeneration; Neuroblastoma; Neurons; Okadaic Acid; Phosphorylation; Rats; tau Proteins; Tumor Cells, Cultured

The effects of okadaic acid (OA), a potent and specific inhibitor of serine/threonine phosphatases 2A and 1, on the transient expression of a human hsp 70 promoter-linked chloramphenicol acetyltransferase gene transfected into N-18 mouse neuroblastoma cells were determined. Assays of reporter gene activity showed that nanomolar concentrations of OA markedly potentiated the heat-induced (but not the basal) expression of pHBCAT, a full-length hsp 70 promoter-driven chloramphenicol acetyltransferase gene construct. This effect of OA was dose-dependent and promoter-specific and appeared to be attributable to inhibition of protein phosphatase 2A as opposed to protein phosphatase 1. The ability of OA to potentiate the heat-induced expression of pHBCAT appeared to be a feature common to several different cell types examined. We propose that the heat-induced transcriptional activation of heat shock genes is associated with the phosphorylation of component(s) of the transcription complex and that OA enhances this phosphorylation, thereby potentiating the heat-induced hsp 70 promoter activity.

Topics: Animals; Base Sequence; Cell Line; Chloramphenicol O-Acetyltransferase; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; HeLa Cells; Hot Temperature; Humans; Kinetics; Mice; Molecular Sequence Data; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Promoter Regions, Genetic; Protein Phosphatase 1; Protein Phosphatase 2; Recombinant Proteins; Transcription, Genetic; Transfection; Tumor Cells, Cultured

When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (OA; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate-dependent NF-H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30%, but did not increase neurite contour length. Ultrastructural analysis demonstrated a > 2-fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down-regulation during dbcAMP-mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate-dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF-H subunits from cytoskeletons of OA-treated cells were less susceptible to degradation by an endogenous calcium-dependent protease, providing a possible mechanism for neurofilament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament-microtubule interaction.

Topics: Animals; Axons; Bucladesine; Ethers, Cyclic; Intermediate Filaments; Microtubules; Neurites; Neuroblastoma; Neurofilament Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.

Topics: Adrenal Gland Neoplasms; Animals; Cell Adhesion; Diglycerides; Ethers, Cyclic; Glioma; Laminin; Neoplasm Proteins; Neuroblastoma; Neurons; Okadaic Acid; Pheochromocytoma; Phorbol Esters; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Tumor Cells, Cultured; Vanadates