okadaic-acid and Nerve-Degeneration

Okadaic acid (OKA) is one of the main polyether toxins produced by marine microalgae which causes diarrhetic shellfish poisoning. It is a selective and potent inhibitor of serine/threonine phosphatases 1 and 2A induces hyperphosphorylation of tau in vitro and in vivo. The reduced activity of phosphatases like, protein phosphatase 2A (PP2A) has been implicated in the brain of Alzheimer's disease (AD) patients. It is reported that AD is a complex multifactorial neurodegenerative disorder and hyperphosphorylated tau proteins is a major pathological hallmark of AD. The molecular pathogenesis of AD includes an extracellular deposition of beta amyloid (Aβ), accumulation of intracellular neurofibrillary tangles (NFT), GSK3β activation, oxidative stress, altered neurotransmitter and inflammatory cascades. Several lines of evidence suggested that the microinfusion of OKA into the rat brain causes cognitive deficiency, NFTs-like pathological changes and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases. So, communal data and information inferred that OKA induces neurodegeneration along with tau hyperphosphorylation; GSK3β activation, oxidative stress, neuroinflammation and neurotoxicity which is a characteristic feature of AD pathology. Through this collected evidence, it is suggested that OKA induced neurotoxicity may be a novel tool to study Alzheimer's disease pathology and helpful in development of new therapeutic approach.

Topics: Alzheimer Disease; Animals; Brain; Disease Models, Animal; Humans; Nerve Degeneration; Neurotoxicity Syndromes; Okadaic Acid; Rats

This communication presents a novel experimental model for Alzheimer studies, where connected primary neurons were set into subtend, co-pathological states. Cortical neurons were cultured in two separated cell compartments in a microfluidic device. A neurite network was generated in a main channel through the neurite outgrowth from both cell compartments. A gradient of okadaic acid (OA) is generated over this neurite network by perfusion. OA is a phosphatase inhibitor that induces hyperphosphorylation of Tau proteins, a major hallmark in Alzheimer disease. The local OA treatment resulted in a connected "diseased" and "healthy" cell population. Anti-phosphorylated tau (Ser262) staining confirmed different states of phosphorylated Tau proteins, and synapthophysin staining the connection of "healthy" and "diseased" cells. Here, we present a novel in vitro model that opens the possibility to study cellular and molecular propagation mechanisms in neurodegeneration, in Tauopathies (as e.g., in Alzheimer), as well as simultaneous drug effects on connected healthy and diseased cell populations.

Topics: Alzheimer Disease; Animals; Cells, Cultured; Cerebral Cortex; Cytological Techniques; Microfluidic Analytical Techniques; Models, Neurological; Nerve Degeneration; Neurons; Okadaic Acid; Phosphorylation; Rats; Rats, Wistar; tau Proteins

Phosphorylation of eukaryotic initiation factor-2 alpha (eIF2 alpha) is increased in Alzheimer's disease (AD) and this protein can be phosphorylated by several kinases, including double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), amino acids-regulated eIF2 alpha kinase (GCN2) and heme-regulated eIF2 alpha kinase (HRI). PKR and PERK especially are activated in the AD brain, and GCN2 is reported to increase presenilin-1 (PS1) activity. Okadaic acid (OA), a protein phosphatase-2A (PP2A) inhibitor, is known to increase tau phosphorylation, beta-amyloid (A beta) deposition and neuronal death, which are the pathological characteristics of AD. Here, we show that the phosphorylation of eIF2 alpha is increased and its kinases, PKR, PERK and GCN2 are activated in rat neurons by OA. Activating transcription factor (ATF4) which induces apoptosis in response to eIF2 alpha phosphorylation was increased and translocated to nuclei in OA-treated neurons. These results suggest that the successive events of activation of eIF2 alpha kinases and eIF2 alpha phosphorylation leading to ATF4 nuclear translocation may contribute to neuronal death. However, PKR inhibitors did not reduce eIF2 alpha phosphorylation or neuronal toxicity despite inhibiting PKR activity. These results suggest that PKR might not be the most responsible kinase for eIF2 alpha phosphorylation or cell death in PP2A-inhibited conditions such as AD.

Topics: Animals; Cells, Cultured; eIF-2 Kinase; Enzyme Activation; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Nerve Degeneration; Neurons; Okadaic Acid; Protein Kinases; Rats

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

Treatment of neurons with okadaic acid, a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death, and to create a research model of Alzheimer's disease. Amyloid precursor protein (APP) is the precursor protein of the beta-amyloid peptide that accumulates in extracellular plaques in Alzheimer's disease. Several studies have shown that mint-1 (munc18-interacting protein 1) and mint-2 bind to the YENPTY motif in the cytoplasmic domain of APP and inhibit processing of APP to beta-amyloid peptide. Here, we report that, upon neurodegeneration with okadaic acid, mint-1 and mint-2 levels were reduced by proteolytic cleavage, and that these changes were followed by increases in APP levels. We also show that the mint-1 and mint-2 cleavage and APP overexpression were prevented by calpain inhibitor-I and inhibitor-II. These results indicate that mint cleavage might play a role in the pathophysiology of Alzheimer's disease.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cadherins; Calpain; Carrier Proteins; Cells, Cultured; Cerebral Cortex; Down-Regulation; Enzyme Inhibitors; Glycoproteins; Membrane Proteins; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Okadaic Acid; Oligopeptides; Rats

Apoptosis via tau phosphorylation has been implicated in the selective neuronal losses seen in Alzheimer's disease (AD). Previous studies in vivo and in cultured neurons have shown that okadaic acid (OA) evokes tau phosphorylation to initiate a neurodegeneration that resembles the pathogenesis of AD. In an effort to identify additional key molecules in this neurodegeneration, we treated cultured rat neurons with OA and examined the apoptosis-related effects, such as changes in mitochondrial activity and expression levels of JNK, Bim, Bad, Bax and caspase-3. Western blotting revealed that phosphorylation of JNK and c-jun occurred first, followed by increased expression of Bim and subsequent caspase-3 activation in OA-treated neurons. In contrast, Bad levels decreased as early as 4 h after OA treatment. Immunocytochemistry showed that the increased phospho-JNK immunoreactivity was localized in the cytosol of degenerating neurons, while increased phospho-c-jun was localized in the nucleus. The mitochondria showed decreased membrane potential and increased swelling after OA treatment. Collectively, these data suggest that JNK- and Bim-related mitochondrial dysfunction is involved in OA-induced neurodegeneration.

Topics: Animals; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; Blotting, Western; Caspase 3; Caspases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; MAP Kinase Kinase 4; Membrane Potentials; Membrane Proteins; Mitochondria; Nerve Degeneration; Neurons; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins; Rats

Emerging evidence suggests that not only beta-amyloid but also other amyloid precursor protein (APP) fragments, such as the beta-C-terminal fragment (betaCTF), might be involved in Alzheimer's disease (AD). Treatment of neurons with okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death to create a research model of AD. In this study, we analyzed axonopathy and APP regulation in cultured rat neurons treated with OA. After OA treatment, the neurons presented with axonal swellings filled with vesicles, microtubule fragments, and transport molecules such as kinesin and synapsin-I. Western blotting showed that intracellular APP levels were increased and immunocytochemistry using antibodies against the APP C-terminus showed that APP accumulated in the axonal swellings. This APP C-terminus immunoreactivity disappeared when neurons were cotreated with a beta-secretase inhibitor, but not with alpha- or gamma-secretase inhibitors, indicating that the accumulation was primarily composed of APP-betaCTF. These findings provide the first evidence that APP-betaCTF can accumulate in the axons of OA-treated neurons, and may suggest that APP-betaCTF is involved in the pathogenesis of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Axonal Transport; Cells, Cultured; Cytoprotection; Disease Models, Animal; Down-Regulation; Endopeptidases; Enzyme Inhibitors; Microscopy, Electron, Transmission; Nerve Degeneration; Okadaic Acid; Peptide Fragments; Presynaptic Terminals; Rats

Calpain activation has been implicated in the pathogenesis of Alzheimer's disease. Okadaic acid, a protein phosphatase-2A inhibitor, has been used in Alzheimer's disease research models to increase tau phosphorylation and induce neuronal death. We previously reported that okadaic acid induced predominant activation of caspase-3 in immature neurons, but less activation in mature neurons. We found here that, in okadaic-acid-treated mature neurons, levels of an inactive form of m-calpain decreased and levels of calpain-cleaved spectrin and synapsin-I fragments increased, suggestive of calpain activation. Pretreatment with calpain inhibitor decreased lactate dehydrogenase release by 20% and increased average dendritic branch length by 50% compared with neurons treated with okadaic acid alone. These findings suggest that calpain is activated during okadaic-acid-induced neurodegeneration and calpain inhibition can be protective against it.

Topics: Animals; Blotting, Western; Calpain; Caspase 3; Caspases; Cells, Cultured; Cerebral Cortex; Drug Interactions; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Glycoproteins; L-Lactate Dehydrogenase; Microtubule-Associated Proteins; Nerve Degeneration; Neurons; Okadaic Acid; Rats

Hyperphosphorylation of tau is a characteristic feature of the neurodegenerative pathology in Alzheimer's disease (AD). Okadaic acid is used as a research model of AD to increase the tau phosphorylation and neuronal death. Using Western blotting, we found that the amounts of activated PKB[pS-473] and inactivated GSK-3beta[pS-9] were increased in proportion to the progress of okadaic acid induced tau phosphorylation. Immunocytochemistry showed that PKB[pS-473] and GSK-3beta[pS-9] immunoreactivity increased in dystrophic neurites and cell bodies in degenerating neurons after okadaic acid treatment. Double staining with phosphospecific tau antibodies showed that PKB[pS-473] and GSK-3beta[pS-9] were colocalized with phosphospecific tau in response to okadaic acid. Taken together, our data suggest that inhibition of protein phosphatase results in the hyperphosphorylation of tau without GSK-3beta overactivation.

Topics: Animals; Blotting, Western; Cells, Cultured; Cerebral Cortex; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunohistochemistry; Nerve Degeneration; Neurons; Okadaic Acid; Oncogene Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; tau Proteins; Time Factors

Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, reduces the clinical deterioration in moderate-to-severe Alzheimer disease (AD) for which other treatments are not available. The activity of protein phosphatase (PP)-2A is compromised in AD brain and is believed to be a cause of the abnormal hyperphosphorylation of tau and the consequent neurofibrillary degeneration. Here we show that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices. Such restorative effects of memantine were not detected either with 5,7-dichlorokynurenic acid or with D(-)-2-amino-5-phosphopentanoic acid, NMDA receptor antagonists active at the glycine binding site and at the glutamate binding site, respectively. These findings show (1) that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation of tau/neurofibrillary degeneration and (2) that this drug might be useful for the treatment of AD and related tauopathies.

Topics: 2-Amino-5-phosphonovalerate; Alzheimer Disease; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Kynurenic Acid; Memantine; Microtubule-Associated Proteins; Nerve Degeneration; Neurofibrils; Neuroprotective Agents; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Wistar; tau Proteins

An imbalanced phosphorylation system is recognized to be one of the main reasons for Alzheimer-like hyperphosphorylation of cytoskeletal proteins. However, little is known about the strategies rectifying the lesions caused by this disrupted phosphorylation. To search for the means to arrest Alzheimer-like damages and explore the underlying mechanisms, in this study we treated N2a/peuht40 cells with okadaic acid (OA), a specific inhibitor of protein phosphatase-2A (PP-2A) and PP-1, to mimic an Alzheimer-like phosphatase-deficient system and then used heat preconditioning (42 degrees C for 1 hour) to induce the expression of inducible heat shock protein 70 (Hsp70) in the cells. We observed that heat preconditioning arrested OA-induced hyperphosphorylation of neurofilament (NF) protein at SMI34 and SMI33 epitopes as well as hyperphosphorylation of tau at Tau-1 and PHF-1 epitopes. It counteracted OA-induced decrease in PP-2A activity with a concurrent inhibition in constitutive activity of mitogen-activated protein kinases (MAPKs) and cyclic adenosine 5'-monophosphate-dependent protein kinase A (PKA). Conversely, quercetin, a recognized blocker of stress-responsive Hsp70 expression, diminished the effects caused by heat preconditioning. These results suggested that Hsp70 antagonized OA-induced Alzheimer-like NF and tau hyperphosphorylation, and the restoration of PP-2A and inhibition of MAPKs-PKA activity might be part of the underlying mechanisms for the rectification of OA-induced hyperphosphorylation.

Topics: Alzheimer Disease; Antibodies, Monoclonal; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Enzyme Inhibitors; Heat-Shock Response; HSP70 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; Nerve Degeneration; Neurofilament Proteins; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Quercetin; tau Proteins; Tumor Cells, Cultured

We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.

Topics: Anesthetics, Inhalation; Animals; Electroencephalography; Epilepsy; Extracellular Fluid; Glutamic Acid; Halothane; Hippocampus; Male; Microdialysis; Nerve Degeneration; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Wistar; Wakefulness

We investigated the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, on spatial memory and neuronal survival in rats. Rats were initially trained on a spatial memory task in an eight arm radial maze. Spatial reference and working memory was impaired 1 day after the unilateral microinjection of OA into the dorsal hippocampus. The impairment was transient, and had disappeared by the following day. In contrast, neurodegeneration induced by OA was persistent and extended to the contralateral side 13 days after the injection. These results suggest that OA causes spatial memory impairment and neurodegeneration when injected directly into the hippocampus. Our findings also indicate dissociation between memory impairment and neurodegeneration induced by OA.

Topics: Animals; Cell Count; Enzyme Inhibitors; Hippocampus; Male; Maze Learning; Memory; Memory Disorders; Mitogen-Activated Protein Kinases; Nerve Degeneration; Neurons; Neurotoxins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Pyramidal Cells; Rats; Rats, Wistar; Space Perception

Peroxidation of membrane lipids occurs in many different neurodegenerative conditions including stroke, and Alzheimer's and Parkinson's diseases. Recent findings suggest that lipid peroxidation can promote neuronal death by a mechanism involving production of the toxic aldehyde 4-hydroxy-2,3-nonenal (HNE), which may act by covalently modifying proteins and impairing their function. The transcription factor NF-kappa B can prevent neuronal death in experimental models of neurodegenerative disorders by inducing the expression of anti-apoptotic proteins including Bcl-2 and manganese superoxide dismutase. We now report that HNE selectively suppresses basal and inducible NF-kappa B DNA binding activity in cultured rat cortical neurons. Immunoprecipitation-immunoblot analyses using antibodies against HNE-conjugated proteins and p50 and p65 NF-kappa B subunits indicate that HNE does not directly modify NF-kappa B proteins. Moreover, HNE did not affect NF-kappa B DNA-binding activity when added directly to cytosolic extracts, suggesting that HNE inhibits an upstream component of the NF-kappa B signaling pathway. Inhibition of the survival-promoting NF-kappa B signaling pathway by HNE may contribute to neuronal death under conditions in which membrane lipid peroxidation occurs.

Topics: Aldehydes; Alzheimer Disease; Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Lipid Peroxidation; Nerve Degeneration; Neurons; NF-kappa B; Okadaic Acid; Protein Synthesis Inhibitors; Rats; Stroke; Transcription Factor AP-1; Vanadates

Within neurofibrillary tangles and dystrophic neurites of Alzheimer's disease (AD), the cytoskeletal protein tau is abnormally hyperphosphorylated. In the present study, we examined the effect of okadaic acid (OA), a protein phosphatase inhibitor, in rat cultured neurons. Low concentrations of OA induce degeneration of neurites, rounding of cell bodies, detachment from the substratum, and eventual neuronal death. During OA-induced degeneration, SMI-31 immunoreactivity became punctate in neurites at 6 h after OA treatment, and over time, accumulated in cell bodies and dystrophic neurites. Hyperphosphorylation of tau and marked loss of MAP-2-positive dendrites occurred after 6 h of treatment with OA. Thereafter, AT-8 and PHF-1 immunoreactivity accumulated in cell bodies and subsequently appeared in distal axon-like neurites. These results demonstrate that OA treatment induced hyperphosphorylation of tau and preferential dendritic damage, with subsequent accumulation of phosphorylated tau in cell bodies and dystrophic axon-like neurites. OA-induced neurodegeneration may provide a useful model to study AD.

Topics: Animals; Antibodies, Monoclonal; Axons; Cell Death; Cell Size; Cells, Cultured; Dendrites; Enzyme Inhibitors; Fetus; Hippocampus; L-Lactate Dehydrogenase; Microtubule-Associated Proteins; Nerve Degeneration; Neurofibrillary Tangles; Neurons; Okadaic Acid; Phosphorylation; Rats; Rats, Sprague-Dawley; tau Proteins

The regional selectivity and mechanisms underlying the toxicity of the serine/threonine protein phosphatase inhibitor okadaic acid (OA) were investigated in hippocampal slice cultures. Image analysis of propidium iodide-labeled cultures revealed that okadaic acid caused a dose- and time-dependent injury to hippocampal neurons. Pyramidal cells in the CA3 region and granule cells in the dentate gyrus were much more sensitive to okadaic acid than the pyramidal cells in the CA1 region. Electron microscopy revealed ultrastructural changes in the pyramidal cells that were not consistent with an apoptotic process. Treatment with okadaic acid led to a rapid and sustained tyrosine phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2 (p44/42(mapk)). The phosphorylation was markedly reduced after treatment of the cultures with the microbial alkaloid K-252a (a nonselective protein kinase inhibitor) or the MAP kinase kinase (MEK1/2) inhibitor PD98059. K-252a and PD98059 also ameliorated the okadaic acid-induced cell death. Inhibitors of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, or tyrosine kinase were ineffective. These results indicate that sustained activation of the MAP kinase pathway, as seen after e.g., ischemia, may selectively harm specific subsets of neurons. The susceptibility to MAP kinase activation of the CA3 pyramidal cells and dentate granule cells may provide insight into the observed relationship between cerebral ischemia and dementia in Alzheimer's disease.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antioxidants; Apoptosis; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Enzyme Inhibitors; Flavanones; Flavonoids; Genistein; Hippocampus; Indole Alkaloids; Male; Microscopy, Electron; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nerve Degeneration; Neurons; Okadaic Acid; Organ Culture Techniques; Phosphoric Monoester Hydrolases; Propidium; Protein Kinase Inhibitors; Protein Kinases; Rats; Rats, Wistar; Staurosporine; Sulfonamides

The tumor promoter okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A and therefore it is a useful tool for studying the participation of protein phosphorylation in cellular processes. Since it has been shown that in cultured neurons OKA behaves as a potent neurotoxin, in the present work we have administered different doses of this compound into the dorsal rat hippocampus, in order to assess its neurotoxicity in vivo. Cresyl violet staining of hippocampal sections revealed that as early as 3 h after injection of 300 ng OKA a notable neurodegeneration occurred in the CA1 subfield, the dentate gyrus, and the hilus, particularly in the former. Neuronal death was more evident at 24 h and at this time the extent of damage was dose-dependent. The process of neuronal death was accompanied by a loss of the microtubule-associated protein MAP2, as assessed by immunocytochemistry. Moreover, OKA treatment resulted in a notable expression of the inducible heat shock protein 72 in the surviving neurons of the injected hippocampus and in the corresponding CA1 and hilus of the apparently normal contralateral hippocampus. The expression of the heat shock protein was partially prevented in the injected hippocampus and completely blocked in the contralateral CA1 region, by the systemic previous administration of the NMDA receptor antagonist MK-801. These results suggest that protein hyperphosphorylation due to inhibition of phosphatases in vivo induces neuronal stress and subsequent neurodegeneration.

Topics: Animals; Dentate Gyrus; Dizocilpine Maleate; Enzyme Inhibitors; Functional Laterality; Gene Expression Regulation; Heat-Shock Proteins; Hippocampus; Immunohistochemistry; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Wistar

We have previously described the marine toxin okadaic acid (OKA) to be a potent neurotoxin for cultured rat cerebellar neurons. Here we show that OKA-induced neurodegeneration involves the DNA fragmentation characteristic of apoptosis and is protein synthesis-dependent. DNA fragmentation and neurotoxicity correlated with inhibition of protein phosphatase (PP) 2A rather than PP1 activity. Neurotrophins NT-3 and BDNF failed to protect from OKA-induced apoptotic neurotoxicity that was, however, totally prevented by insulin-like growth factor-1. Neuronal death by OKA was significantly reduced by protein kinase C inhibitors and by the L-type calcium channel agonist Bay K8644, while it was potentiated by the reduction of free extracellular calcium concentrations.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Calcium Channel Agonists; Cells, Cultured; Cerebellum; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gene Expression Regulation; Insulin-Like Growth Factor I; Nerve Degeneration; Nerve Growth Factors; Neurons; Neurotoxins; Neurotrophin 3; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Rats

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