okadaic-acid and Alzheimer-Disease

This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in β-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer's disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce β-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Dinoflagellida; Marine Toxins; Okadaic Acid; Plaque, Amyloid; tau Proteins; Zebrafish

Okadaic acid (OKA), a polyether C38 fatty acid toxin extracted from a black sponge Hallichondria okadaii, is a potent and selective inhibitor of protein phosphatase, PP1 and PP2A. OKA has been proved to be a powerful probe for studying the various regulatory mechanisms and neurotoxicity. Because of its property to inhibit phosphatase activity, OKA is associated with protein phosphorylation; it is implicated in hyperphosphorylation of tau and in later stages causes Alzhiemer's disease (AD)-like pathology. AD is a progressive neurodegenerative disorder, pathologically characterized by extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). The density of tau tangles in AD pathology is associated with cognitive dysfunction. Recent studies have highlighted the importance of serine/threonine protein phosphatases in many processes including apoptosis and neurotoxicity. Although OKA causes neurotoxicity by various pathways, the exact mechanism is still not clear. The activation of major kinases, such as Ser/Thr, MAPK, ERK, PKA, JNK, PKC, CaMKII, Calpain, and GSK3β, in neurons is associated with AD pathology. These kinases, associated with abnormal hyperphosphorylation of tau, suggest that the cascade of these kinases could exclusively be involved in the pathogenesis of AD. The activity of serine/threonine protein phosphatases needs extensive study as these enzymes are potential targets for novel therapeutics with applications in many diseases including cancer, inflammatory diseases, and neurodegeneration. There is a need to pay ample attention on MAPK kinase pathways in AD, and OKA can be a better tool to study cellular and molecular mechanism for AD pathology. This review elucidates the regulatory mechanism of PP2A and MAPK kinase and their possible mechanisms involved in OKA-induced apoptosis, neurotoxicity, and AD-like pathology.

Topics: Alzheimer Disease; Animals; Humans; Neurons; Neurotoxins; Okadaic Acid; Phosphorylation; Signal Transduction

The molecular pathogenesis of Alzheimer's disease (AD) includes a variety of risk factors, extracellular deposition of β-amyloid, accumulation of intracellular neurofibrillary tangles, oxidative neuronal damage and inflammatory cascades. Although amyloid-β-containing senile plaques and phospho-tau-containing neurofibrillary tangles are hallmark lesions of AD, neither is specific to nor even a marker of the disease. From a biochemical point of view the most consistent finding is a decreased level of choline acetyltransferase. In recent years, cumulative evidence has been gained on the involvement of neuronal lipoprotein activity, and on the role of cholesterol and other lipids in pathogenesis. Although basic research has made remarkable progress in the past two decades, currently available drugs are only able to improve cognitive symptoms temporarily and no treatment can reverse, stop or even slow this inexorable neurodegenerative process.. The various neurobiological events associated with development of AD and the multiple treatment approaches for combating this disorder.. AD is a complex multifactorial disorder and thus a single target or pathogenic pathway is unlikely to be identified. Developing therapeutic interventions demands a greater understanding of the processes and the differential involvement of the various mediators. Effective therapeutics are urgently needed, and it is hoped that anti-amyloid strategies will offer a significant step towards a causal therapy.

Topics: Aluminum; Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Inflammation; Mitochondria; Okadaic Acid; Oxidative Stress; tau Proteins

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by tau aggregating into neurofibrillary tangles. Targeting tau aggregation is one of the most critical strategies for AD treatment and prevention. Herein, a high-throughput screening of tau-aggregation inhibitors was performed by thioflavin T (ThT) fluorescence assay and tauR3 peptides. According to bioactivity-guided isolation, homoprejadomycin (1) was obtained from the marine bacterium Streptomyces tendae MCCC 1A01534. Two new stable derivatives, 2 and 3, were yielded in a one-step reaction. By ThT assay, transmission electron microscopy, and circular dichroism, we demonstrated that the angucyclinones 2 and 3 inhibited tau aggregation and disaggregated tau fibrils. In the presence of 2, native tauR3 peptides maintained the disorder conformation, whereas the tauR3 aggregates reduced β-sheet structures. And compound 2 was confirmed to inhibit the aggregation of full-length 2N4R tau protein. Furthermore, 2 with low cytotoxicity protected HT22 cells from okadaic acid-induced damage by suppressing tau aggregates. These results indicated that 2 was a promising lead structure with tau therapeutic potency for AD treatment.

Topics: Alzheimer Disease; Humans; Neurodegenerative Diseases; Okadaic Acid; Peptides; tau Proteins

Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient activity of PP2A is the responsible of severe pathologies. For instance, one of the main histopathological features of Alzheimer's disease is neurofibrillary tangles, which are mainly comprised by hyperphosphorylated forms of tau protein. This altered rate of tau phosphorylation has been correlated with PP2A depression AD patients. With the goal of preventing PP2A inactivation in neurodegeneration scenarios, we have aimed to design, synthesize and evaluate new ligands of PP2A capable of preventing its inhibition. To achieve this goal, the new PP2A ligands present structural similarities with the central fragment C19-C27 of the well-established PP2A inhibitor okadaic acid (OA). Indeed, this central moiety of OA does not exert inhibitory actions. Hence, these compounds lack PP2A-inhibiting structural motifs but, in contrast, compete with PP2A inhibitors, thus recovering phosphatase activity. Proving this hypothesis, most compounds showed a good neuroprotective profile in neurodegeneration models related to PP2A impairment, highlighting derivative 10, named ITH12711, as the most promising one. This compound (1) restored in vitro and cellular PP2A catalytic activity, measured on a phospho-peptide substrate and by western-blot analyses, (2) proved good brain penetration measured by PAMPA, and (3) prevented LPS-induced memory impairment of mice in the object recognition test. Thus, the promising outcomes of the compound 10 validate our rational approach to design new PP2A-activating drugs based on OA central fragment.

Topics: Alzheimer Disease; Animals; Mice; Neuroprotection; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; tau Proteins; Tauopathies

Since Alzheimer's disease (AD) is a complex and multifactorial neuropathology, the discovery of multi-targeted inhibitors has gradually demonstrated greater therapeutic potential. Neurofibrillary tangles (NFTs), the main neuropathologic hallmarks of AD, are mainly associated with hyperphosphorylation of the microtubule-associated protein Tau. The overexpression of GSK3β and DYRK1A has been recognized as an important contributor to hyperphosphorylation of Tau, leading to the strategy of using dual-targets inhibitors for the treatment of this disorder. ZDWX-12 and ZDWX-25, as harmine derivatives, were found good inhibition on dual targets in our previous study. Here, we firstly evaluated the inhibition effect of Tau hyperphosphorylation using two compounds by HEK293-Tau P301L cell-based model and okadaic acid (OKA)-induced mouse model. We found that ZDWX-25 was more effective than ZDWX-12. Then, based on comprehensively investigations on ZDWX-25 in vitro and in vivo, 1) the capability of ZDWX-25 to show a reduction in phosphorylation of multiple Tau epitopes in OKA-induced neurodegeneration cell models, and 2) the effect of reduction on NFTs by 3xTg-AD mouse model under administration of ZDWX-25, an orally bioavailable, brain-penetrant dual-targets inhibitor with low toxicity. Our data highlight that ZDWX-25 is a promising drug for treating AD.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Mice; Okadaic Acid; Phosphorylation; tau Proteins

The presence of Transient Receptor Potential Vanilloid 1 (TRPV1) channels was detected in many regions of the human and rat brain, including the cortex and hippocampus. TRPV1 channels have functions such as the modulation of synaptic transmission and plasticity and the regulation of cognitive functions. Previous studies conducted with TRPV1 agonists and antagonists show that this channel is associated with the neurodegenerative process. In the present study, the purpose was to investigate the effects of capsaicin, which is a TRPV1 agonist, and capsazepine, a TRPV1 antagonist, in the Alzheimer's Disease (AD) model that was induced by intracerebroventricular (ICV) administration of okadaic acid (OKA).. The AD-like experimental model was created with bilateral ICV OKA injection. Intraperitoneal capsaicin and capsazepine injections were administered to the treatment groups for 13 days and histological and immunohistochemical examinations were performed from the cortex and hippocampal CA3 regions of the brain. The Morris Water Maze Test was used for spatial memory measurement.. ICV OKA administration increased the levels of caspase-3, phosphorylated-tau-(ser396), Aβ, TNF-α, and IL1-β, from the cortex and hippocampal CA3 regions of the brain and decreased the phosphorylated-Glycogen synthase kinase-3 beta-(ser9) levels. In addition, the OKA administration corrupted the spatial memory. The TRPV1 agonist capsaicin reversed the pathological changes induced by ICV OKA administration, but not the TRPV1 antagonist capsazepine.. It was found in the study that the administration of the TRPV1 agonist capsaicin reduced neurodegeneration, neuroinflammation, and deterioration in spatial memory in the AD model induced by OKA.

Topics: Alzheimer Disease; Animals; Antineoplastic Agents; Capsaicin; Humans; Neuroprotective Agents; Okadaic Acid; Rats; TRPV Cation Channels

Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's disease (AD). Despite having multiple treatment options that can mitigate AD symptoms, there is an imperative call to advance our understanding of the disease pathogenesis to unfold disease-modifying treatments/therapies. To explore the driving mechanisms of AD development, we stretch out further to study time-dependant changes after Okadaic acid (OKA)-induced AD-like conditions in zebrafish. We evaluated the pharmacodynamics of OKA at two-time points, i.e., after 4-days and 10-days exposure to zebrafish. T-Maze was utilized to observe the learning and cognitive behaviour, and inflammatory gene expressions such as 5-Lox, Gfap, Actin, APP, and Mapt were performed in zebrafish brains. To scoop everything out from the brain tissue, protein profiling was performed using LCMS/MS. Both time course OKA-induced AD models have shown significant memory impairment, as evident from T-Maze. Gene expression studies of both groups have reported an overexpression of 5-Lox, GFAP, Actin, APP, and OKA 10D group has shown remarkable upregulation of Mapt in zebrafish brains. In the case of protein expression, the heatmap suggested an important role of some common proteins identified in both groups, which can be explored further to investigate their mechanism in OKA-induced AD pathology. Presently, the preclinical models available to understand AD-like conditions are not completely understood. Hence, utilizing OKA in the zebrafish model can be of great importance in understanding the pathology of AD progression and as a screening tool for drug discovery.

Topics: Actins; Aged; Alzheimer Disease; Animals; Brain; Disease Models, Animal; Genomics; Humans; Okadaic Acid; Proteomics; Zebrafish

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

The activation of microglia is found to be associated with neurodegenerative disorders including Alzheimer's disease (AD). Several studies have shown that okadaic acid (OA) induced deposition of tau hyperphosphorylation, and subsequent neuronal degeneration, loss of synapses, and memory impairment, all of which resemble the pathology of AD. Although OA is a powerful tool available for mechanisms of the neurotoxicity associated with AD, the exact mechanism underlying the activation of microglial cells remains unrevealed. The aim of this study was to determine the effect of both OA and OA-treated neuroblastoma SH-SY5Y cells on microglial HAPI cell viability, activation, and phagocytosis. The results showed that both OA and OA-treated neurons did not induce any detectable cytotoxicity of microglial cells. Furthermore, incubation with OA-treated SH-SY5Y cells could increase the expression of ionized calcium-binding adapter molecule 1 (Iba1) on microglial HAPI cells. This result indicated that OA may induce microglial activation through the toxicity of neurons. Moreover, we also demonstrated that OA-treated SH-SY5Y cells were engulfed by CD11b/c-labeled microglial HAPI cells, which were abolished after treatment with 10 mM O-phospho- l-serine ( L-SOP) for 30 min before co-culture with OA-treated SH-SY5Y cells, indicating cells experiencing phagocytic activity. We also confirmed that OA treatment for 24 h significantly increased tau hyperphosphorylation at S396 in SH-SY5Y cells. In conclusion, our findings indicate that OA is a potential toxic inducer underlying the role of microglia in AD pathogenesis.

Topics: Alzheimer Disease; Cell Line, Tumor; Humans; Microglia; Okadaic Acid; Phagocytosis

Topics: Alzheimer Disease; Amyloid beta-Peptides; Glycogen Synthase Kinase 3 beta; Humans; Okadaic Acid; Phenanthrenes; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Synaptophysin; tau Proteins

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

Alzheimer's disease (AD) is a progressive, fatal, neurodegenerative disorder for which only treatments of limited efficacy are available. Despite early mentions of dementia in the ancient literature and the first patient diagnosed in 1906, the underlying causes of AD are not well understood. This study examined the possible role of dopamine, a neurotransmitter that is involved in cognitive and motor function, in AD. We treated adult zebrafish (

Topics: Alzheimer Disease; Animals; Carbon Fiber; Dopamine; Microelectrodes; Neurotransmitter Agents; Okadaic Acid; Zebrafish

Many elderly individuals suffer from Alzheimer's disease (AD), which causes a growing concern. We investigated the mechanism underlying the effects of vitamin D (VD) as a prophylactic treatment. A mouse model of okadaic-acid-induced AD-like pathology was used

Topics: Aged; Alzheimer Disease; Cognitive Dysfunction; Humans; Okadaic Acid; Phosphorylation; tau Proteins; Vitamin D

Alzheimer's disease (AD) is the most common neurodegenerative disease, and it manifests as progressive memory loss and cognitive decline. However, there are no effective therapies for AD, which is an urgent problem to solve. Evodiamine, one of the main bioactive ingredients of. A protein phosphatase 2A inhibitor, okadaic acid (OA), was used to induce tau phosphorylation to mimic AD-like models in neuronal cells. Protein expression and cell apoptosis were detected using Western blotting and flow cytometry, respectively. Spatial memory/cognition was assessed using water maze, passive avoidance tests, and magnetic resonance imaging assay in OA-induced mice models, and brain slices were evaluated further by immunohistochemistry.. The results showed that evodiamine significantly reduced the expression of phosphor-tau, and further decreased tau aggregation and neuronal cell death in response to OA treatment. This inhibition was found to be via the inhibition of glycogen synthase kinase 3β, cyclin-dependent kinase 5, and mitogen-activated protein kinase pathways. In vivo results indicated that evodiamine treatment ameliorated learning and memory impairments in mice, whereas Western blotting and immunohistochemical analysis of the mouse brain also confirmed the neuroprotective effects of evodiamine.. Evodiamine can decrease the neurotoxicity of tau aggregation and exhibit a neuroprotective effect. Our results demonstrate that evodiamine has a therapeutic potential for AD treatment.

Topics: Alzheimer Disease; Animals; Apoptosis; Brain; Cell Line; Cognition; Cognition Disorders; Disease Models, Animal; Humans; Male; Maze Learning; Mice; Mice, Inbred ICR; Neurodegenerative Diseases; Neurons; Neuroprotection; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Quinazolines; Spatial Memory; tau Proteins; Tauopathies

Andrographis paniculata (AP) is a native plant with anti-inflammatory and antioxidant properties and used as an official herbal medicine. Recently more and more researches have indicated that AP shows pharmacological effects on Alzheimer's disease (AD) but its mechanism is unclear.. Network pharmacology approach combined with experimental validation was developed to reveal the underlying molecular mechanisms of AP in treating AD.. The compounds of AP from TCM database, the AD-related targets from disease database and the targets corresponding to compounds from swissTargetPrediction were collected. Then DAVID database was used for annotation and enrichment pathways, meanwhile the compound-target, protein-protein interaction from String database and compound-target-pathway network was constructed, molecular modeling was performed using Sybyl-x. Okadaic acid (OKA)-induced cytotoxicity model in PC12 cells was established to verify the mechanism of AP and the key proteins were detected by western blotting.. 28 AP components were identified after ADME filter analysis and 52 targets were gained via mapping predicted targets into AD-related proteins. In addition, after multiple network analysis, the 22 hub target genes were enriched onto pathways involved in AD, such as neuroactive ligand-receptor interaction, serotonergic synapse, Alzheimer's disease, PI3K-Akt and NF-kB signaling pathway. Interestingly, molecular docking simulation revealed that the targets including PTGS2, BACE1, GSK3B and IKBKB had good ability to combine with AP components. Experimental validation in an in vitro system proved that AP treatment obviously increased in levels inactive of p-GSK3β (P < 0.05) and decreased in levels of BACE (P < 0.05), PTGS2 (namely COX2, P < 0.05) and NF-kB protein (P < 0.05) compare with OKA treated group.. Our data provided convincing evidence that the neuroprotective effects of AP might be partially related to their regulation of the APP-BACE1-GSK3B signal axis and inflammation, which should be the focus of study in this field in the future.

Topics: Alzheimer Disease; Andrographis; Animals; Molecular Docking Simulation; Neuroprotective Agents; Okadaic Acid; PC12 Cells; Phytochemicals; Plant Extracts; Protein Interaction Maps; Rats

Mesenchymal stem cells-conditioned medium (MSC-CM) provides a promising cell-free therapy for Alzheimer's disease (AD) mainly due to the paracrine of MSCs, but the precise mechanisms remain unclear. Studies suggests that mitochondrial dysfunction precedes the accumulation of amyloid-β plaques and neurofibrillary tangles, and involves in the onset and development of AD.. In the present study, we evaluated the protective effects and explored the related-mitochondrial mechanisms of human umbilical cord derived MSC-CM (hucMSC-CM) in an AD model in vitro.. To this end, an AD cellular model was firstly established by okadaic acid (OA)-treated SH-SY5Y cells, and then treated by hucMSC-CM to assess the oxidative stress, mitochondrial function, apoptosis, AD-related genes, and signaling pathways.. hucMSC-CM significantly deceased tau phosphorylated at Thr181 (p181-tau) level, which was increased in AD. hucMSC-CM also alleviated intracellular and mitochondrial oxidative stress in OA-treated SH-SY5Y cells. In addition, hucMSC-CM suppressed apoptosis and improved mitochondrial function in OA-treated SH-SY5Y cells. Flow cytometric analysis indicated that hucMSC-CM exerted the protective effects relying on or partly extracellular vesicle (EV) mitochondrial transfer from hucMSCs to OA-treated SH-SY5Y cells. Moreover, RNA sequencing data further demonstrated that hucMSC-CM regulated many AD-related genes, signaling pathways and mitochondrial function.. These results indicated that MSC-CM or MSC-EVs containing abundant mitochondria may provide a novel potential therapeutic approach for AD.

Topics: Alzheimer Disease; Apoptosis; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Electron Transport Complex IV; Enzyme Inhibitors; Extracellular Vesicles; Humans; In Vitro Techniques; Membrane Potential, Mitochondrial; Mesenchymal Stem Cells; Mitochondria; Neurons; Okadaic Acid; Oxidative Stress; Reactive Oxygen Species; tau Proteins; Umbilical Cord

This study aimed to explore effects and mechanisms of 004 (IMM-H004), a novel coumarin derivative, in OKA (okadaic acid)-induced AD (Alzheimer's disease)-like model. In vitro, MTT, LDH, and Annexin V/FITC flow cytometry assay were used to test cell survival. In vivo, OKA microinjection was conducted to simulate AD-like neuropathology. Morris water maze and Nissl staining were used to detect spatial memory function and neuronal damage respectively. Western blot and immunohistochemistry were used to study the mechanisms of 004 in Tau pathology. The results showed that 004 reduced cell death and increased survival in PC12 cells, and decreased neuronal injury in the hippocampus in rats. 004 improved learning and memory functions in OKA-treated rats. The mechanistic studies indicated that 004 inhibited phosphorylation of Tau protein by down-regulating the activity of protein kinases CDK5 and GSK3β and increasing PP2A activity. Overall, 004 improved spatial memory impairments and neuron cells injury induced by OKA; on the other hand, 004 inhibited Tau hyperphosphorylation by regulating CDK5, GSK3β and PP2A.

Topics: Alzheimer Disease; Animals; Apoptosis; Blotting, Western; Coumarins; Disease Models, Animal; Flow Cytometry; Male; Maze Learning; Neuroprotective Agents; Okadaic Acid; PC12 Cells; Rats; Rats, Sprague-Dawley; tau Proteins

Glutamatergic dysfunction is posed as a main stage in neurodegenerative disorders such as Alzheimer's disease (AD). Glutamate-mediated excitotoxicity contributes to cognitive dysfunction and cell death in AD. Ceftriaxone (CFT), a well-known upregulator of GLT-1, selectively induces the expression of glutamate transporter-1 (GLT-1) in different brain regions and therefore can be posed as a potential candidate for elimination of glutamate-induced excitotoxicity which is an early prominent event in AD brains. This study was designed to investigate the electrophysiological and behavioral effects of the β-lactam antibiotic ceftriaxone in okadaic acid (OKA)-induced model of AD.. Male Wistar rats divided into four control, ceftriaxone (CFT), OKA, and OKA plus ceftriaxone (OKA + CFT) groups. OKA was injected intracerebroventricularly (i.c.v., 200 ng/5 μl) into lateral ventricles and after two weeks the evoked field potential recorded from hippocampal perforant path-DG synapses in order to evaluate the effect of ceftriaxone treatment (200 mg/kg/day, i.p.) on long-term potentiation (LTP) and paired-pulse responses.. Results of this study revealed that ceftriaxone treatment significantly ameliorates the OKA-induced attenuation of field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude following high-frequency stimulation and paired-pulse paradigm indicating its beneficial effects on both short-term and long-term plasticity in these neurons. Ceftriaxone also has an improving effect on OKA-induced impairment in short- and long-term memories evaluated by alternation behavior and passive avoidance tasks in rats.. Therefore, this study suggests that GLT-1 might be a promising therapeutic target for treatment of neurodegenerative disorders such as AD in the future.

Topics: Alzheimer Disease; Animals; Anti-Bacterial Agents; Carcinogens; Ceftriaxone; Dentate Gyrus; Excitatory Postsynaptic Potentials; Hippocampus; Long-Term Potentiation; Male; Neuronal Plasticity; Neurons; Okadaic Acid; Rats; Rats, Wistar; Synapses

Early stages of Alzheimer's disease (AD) are characterized by high phosphorylation of microtubule-associated protein tau, which may result from the downregulation of protein phosphatases.. In order to model phosphatase downregulation and analyze its effect on tau aggregation in vitro, we treated neuroblastoma SH-SY5Y cells with okadaic acid (OA), a protein phosphatase inhibitor, and examined high molecular weight phospho-tau species.. OA treatment led to the appearance of heat-stable protein species with apparent molecular weight around 100 kDa, which were immunoreactive to anti-tau antibodies against phosphorylated Ser202 and Ser396. As these high molecular weight tau-immunoreactive proteins (HMW-TIPs) corresponded to the predicted size of two tau monomers, we considered the possibility that they represent phosphorylation-induced tau oligomers. We attempted to dissociate HMW-TIPs by urea and guanidine, as well as by alkaline phosphatase treatment, but HMW-TIPs were stable under all conditions tested. These characteristics resemble properties of certain sodium dodecyl sulfate (SDS)-resistant tau oligomers from AD brains. The absence of HMW-TIPs detection by anti-total tau antibodies Tau46, CP27 and Tau13 may be a consequence of epitope masking and protein truncation. Alternatively, HMW-TIPs may represent previously unreported phosphoproteins cross-reacting with tau.. Taken together, our data provide a novel characterization of an OA-based cell culture model in which OA induces the appearance of HMW-TIPs. These findings have implications for further studies of tau under the conditions of protein phosphatase downregulation, aiming to explain mechanisms involved in early events leading to AD.

Topics: Alzheimer Disease; Antibodies; Cell Line, Tumor; Enzyme Inhibitors; Humans; Models, Biological; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Radioimmunoprecipitation Assay; tau Proteins

Currently, no treatments exist that are able to directly treat against Alzheimer's disease (AD), and we are facing an inevitable increase in the near future of the amount of patients who will suffer from AD. Most animal models of AD are limited by not being able to recapitulate the entire pathology of AD. Recently an AD model in zebrafish was established by using the protein phosphatase 2A inhibitor, okadaic acid (OKA). Administering OKA to zebrafish was able to recapitulate most of the neuropathology associated with AD. Therefore, providing a drug discovery model for AD that is also time and cost efficient. This study was designed to investigate the effects of GSK3β inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) on this newly developed AD model. Fish were divided into 4 groups and each group received a different treatment. The fish were divided into a control group, a group treated with 1 μM TDZD-8 only, a group treated with 1 μM TDZD-8 + 100 nM OKA, and a group treated with 100 nM OKA only. Administering the GSK3β inhibitor to zebrafish concomitantly with OKA proved to be protective. TDZD-8 treatment reduced the mortality rate, the ratio of active: inactive GSK3β, pTau (Ser199), and restored PP2A activity. This further corroborates the use of GSKβ inhibitors in the treatment against AD and bolsters the use of the OKA-induced AD-like zebrafish model for drug discovery.

Topics: Alzheimer Disease; Animals; Brain; Cognition; Disease Models, Animal; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Neurons; Okadaic Acid; tau Proteins; Thiadiazoles; Zebrafish

Okadaic acid (OA)‑induced neurotoxicity may be considered a novel tool used to study Alzheimer's disease (AD) pathology, and may be helpful in the development of a novel therapeutic approach. It has been reported that galangin inhibits β‑site amyloid precursor protein‑cleaving enzyme 1 expression, which is a key enzyme for amyloid β (Aβ) generation and is a potential drug candidate for AD therapy. However, further studies are required to confirm its neuroprotective effects in other AD models. The present study aimed to explore the neuroprotective effects of galangin on OA‑induced neurotoxicity in PC12 cells. The cells were divided into the following groups: Control group, model group (175 nM OA for 48 h) and galangin groups (0.25, 0.5 and 1 µg/ml). Beclin‑1, phosphorylated (p)‑protein kinase B (Akt), p‑glycogen synthase kinase (GSK)3β and p‑mechanistic target of rapamycin (mTOR) expression was also measured in the following PC12 cell groups: Control group, model group, 3‑methyladenine group (5 nM), rapamycin group (100 nM) and galangin group (1 µg/ml). The levels of β‑secretase, Aβ42 and p‑tau were detected by ELISA, Beclin‑1 expression was examined by immunohistochemistry and the protein expression levels of p‑Akt, p‑mTOR p‑GSK3β, and Beclin‑1 were detected by western blotting. Galangin treatment enhanced cell viability in cells treated with OA, and decreased β‑secretase, Aβ42 and p‑tau levels. In addition, it suppressed Beclin‑1 and p‑GSK3β expression, but promoted p‑Akt and p‑mTOR expression by regulating the Akt/GSK3β/mTOR pathway. These results indicated that galangin protected PC12 cells from OA‑induced cytotoxicity and inhibited autophagy via the Akt/GSK3β/mTOR pathway, thus suggesting that it may be considered a potential therapeutic agent for AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Autophagy; Flavonoids; Glycogen Synthase Kinase 3 beta; Humans; Okadaic Acid; PC12 Cells; Peptide Fragments; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; tau Proteins; TOR Serine-Threonine Kinases

Alzheimer's disease (AD) is the most common cause of progressive decline of memory function in aged humans. To study about a disease mechanism and progression, animal models for the specific disease are needed. For AD, although highly valid animal models exist, none of the existing models recapitulates all aspects of human AD. The pathogenic mechanisms involved in AD are diverse and thus it is difficult to recapitulate human AD in model organisms. Intracerebroventricular (ICV) injection of okadaic acid (OKA), a protein phosphatase 2A (PP2A) inhibitor, in rats causes neurotoxicity associated with neurofibrillary degeneration. However, this model lacks amyloid pathology as observed in AD. We aimed at combining two different treatments and hence producing a better animal model of AD which may mimic most of the neuropathological, neurobehavioral, and neurochemical changes observed in AD. For this, OKA (200 ng) was microinjected bilaterally into the hippocampus of male Wistar rats followed by exposure of same rats to hypoxic conditions (10%) for 3 days. The result of which, the combination model exhibited tau hyperphosphorylation along with Aβ upregulation as evident by western blotting and immunohistochemistry. The observed changes were accompanied with dysfunction of neurotransmitter system, i.e., decreased acetylcholine activity and expression. This combinatorial model also exhibited cognitive deficiency which was assessed by Morris water maze and avoidance tests along with enhanced oxidative stress which is thought to be a major player in AD pathogenesis. Taken together, we established an easily reproducible and reliable rat model for sporadic dementia of Alzheimer's type in rats which allows effective testing of new therapeutic strategies.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Avoidance Learning; Cognitive Dysfunction; Disease Models, Animal; Hippocampus; Hypoxia; Male; Maze Learning; Microinjections; Neurons; Okadaic Acid; Oxidative Stress; Rats, Wistar; Stereotaxic Techniques

Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by aberrant tau protein hyperphosphorylation, which eventually leads to the formation of neurofibrillary tangles. Hyperphosphorylated tau protein is considered as a vital factor in the development of AD and is highly associated with cognitive impairment. Therefore, it is recognized to be a potential therapeutic target. Quercetin (QUE) is a naturally occurring flavonoid compound. In the present study, the inhibitory effect of QUE on okadaic acid (OA)-induced tau protein hyperphosphorylation in HT22 cells was explored. Western blotting results indicated that QUE significantly attenuated OA‑induced tau protein hyperphosphorylation at the Ser396, Ser199, Thr231 and Thr205 sites. Further experiments demonstrated that QUE inhibited the activity of cyclin‑dependent kinase 5 (CDK5), a key enzyme in the regulation of tau protein, and blocked the Ca2+‑calpain‑p25‑CDK5 signaling pathway. These observations indicate the ability of QUE to decrease tau protein hyperphosphorylation and thereby attenuate the associated neuropathology. In conclusion, these results support the potential of QUE as a therapeutic agent for AD and other neurodegenerative tauopathies.

Topics: Alzheimer Disease; Calcium; Calpain; Cognitive Dysfunction; Cyclin-Dependent Kinase 5; Hippocampus; Humans; Nerve Tissue Proteins; Neurons; Okadaic Acid; Phosphorylation; Quercetin; tau Proteins

Okadaic acid (OKA) is a protein phosphatase 2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer's disease (AD). Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1 = negative control, Group #2 = 100 nM OKA, Group #3 = 100 nM OKA +500 μM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB (Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

Topics: Alzheimer Disease; Amino Acids, Sulfur; Animals; Brain; Cells, Cultured; Female; Male; Mice; Neuroprotection; Neuroprotective Agents; Okadaic Acid; Zebrafish

Abnormal phosphorylation of tau, one of the most common symptoms of dementia, has become increasingly important in the study of the etiology and development of Alzheimer's disease. Paeoniflorin, the main bioactive component of herbaceous peony, is a monoterpene glycoside, which has been reported to exert beneficial effects on neurodegenerative disease. However, the effect of paeoniflorin on tauopathies remains ambiguous. SH-SY5Y cells were treated with okadaic acid (OA) for 8 h to induce tau phosphorylation and no cell death was observed. Optical microscopy results showed that paeoniflorin ameliorated okadaic acid induced morphological changes, including cell swelling and synapsis shortening. Western blotting data illustrated that paeoniflorin reversed okadaic acid induced tau hyperphosphorylation, which was enhanced by inhibiting the activities of calpain, Akt and GSK-3β. Transmission electron microscopy results showed that paeoniflorin alone can reduce the number of autophagosomes and stabilize the microtubule structure. In addition, calpastain and paeoniflorin enhance the effect of paeoniflorin on stabilizing microtubules. In addition, calpastain markedly enhanced the effect of paeoniflorin on reversing okadaic acid-lowered fluorescence intensity of both MAP-2 and β III-tubulin, two microtubule-associated proteins. This study shows that paeoniflorin protected SH-SY5Y cells against okadaic acid assault by interfering with the calpain/Akt/GSK-3β-related pathways, in which autophagy might be involved. Besides, paeoniflorin is found to relieve the stress response of the microtubule structure system caused by okadaic acid treatment. The results presented in this study suggest that paeoniflorin potentially plays an important role in tauopathies.

Topics: Alzheimer Disease; Autophagy; Calpain; Cell Line, Tumor; Glucosides; Glycogen Synthase Kinase 3 beta; Humans; Microtubules; Monoterpenes; Neurons; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Proto-Oncogene Proteins c-akt; tau Proteins

Six triterpenic acids were separated and purified from the ethyl acetate extractive fraction of ethanol extracts of Potentilla parvifolia FISCH. using a variety of chromatographic methods. The neuroprotective effects of these triterpenoids were investigated in the present study, in which the okadaic acid induced neurotoxicity in human neuroblastoma SH-SY5Y cells were used as an Alzheimer's disease cell model in vitro. The cell model was established with all trans-retinoic acid (5 µmol/L, 4 d) and okadaic acid (40 nmol/L, 6 h) treatments to induce tau phosphorylation and synaptic atrophy. Subsequently, the neuroprotective effects of these triterpenic acids were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3-6 had the better neuroprotective effects. Furthermore, we tested the level of mitochondrial reactive oxygen species and mitochondrial membrane potential of these compounds in SH-SY5Y cells by flow cytometry technology to investigate the potential neuroprotective mechanism of these compounds. All of the results indicated that maybe the mechanism of compounds 5 and 6 is to protect the cell from mitochondrial oxidative stress injuries.

Topics: Alzheimer Disease; Cell Differentiation; Cell Line, Tumor; Cell Survival; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neuroprotective Agents; Okadaic Acid; Oxidative Stress; Plant Components, Aerial; Potentilla; Reactive Oxygen Species; Tretinoin; Triterpenes

Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer's disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca

Topics: Alkaloids; Alzheimer Disease; Calcium; Calcium Channel Blockers; Cell Line, Tumor; Humans; Indole Alkaloids; Indoles; Molecular Docking Simulation; Molecular Structure; Neurons; Neuroprotective Agents; Okadaic Acid; Piperidines; Protein Phosphatase 2; Structure-Activity Relationship

To develop potent multi-target ligands against Alzheimer's disease (AD), a series of novel bivalent β-carboline derivatives were designed, synthesized, and evaluated. In vitro studies revealed these compounds exhibited good multifunctional activities. In particular, compounds 8f and 8g showed the good selectivity potency on BuChE inhibition (IC

Topics: Acetylcholinesterase; Alzheimer Disease; Amyloid beta-Peptides; Binding Sites; Butyrylcholinesterase; Carbolines; Cell Line, Tumor; Cell Survival; Cholinesterase Inhibitors; Drug Design; Humans; Hydrogen Peroxide; Inhibitory Concentration 50; Molecular Docking Simulation; Neuroprotective Agents; Okadaic Acid; Peptide Fragments; Protein Structure, Tertiary; Structure-Activity Relationship

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC

Topics: Alzheimer Disease; Animals; Blood-Brain Barrier; Central Nervous System Agents; Circular Dichroism; Drug Design; Drug Evaluation, Preclinical; Fluorescence Resonance Energy Transfer; Glycogen Synthase Kinase 3; Hep G2 Cells; Humans; Microscopy, Atomic Force; Molecular Targeted Therapy; Okadaic Acid; Phosphorylation; Protein Kinase Inhibitors; Rats; Structure-Activity Relationship; Swine; tau Proteins; Thiazolidinediones

Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disease (AD) patients. In these patients, acetylcholinesterase inhibitors (AChEI) are the major drug class used for their treatment; however, they present unwanted cholinergic side effects and have provided limited efficacy in clinic. Because combination therapy is being extensively used to treat different pathological diseases such as cancer or acquired immune deficiency syndrome, we posed this study to evaluate if melatonin in combination with an AChEI, galantamine, could provide beneficial properties in a novel in vitro model of AD. Thus, we subjected organotypic hippocampal cultures (OHCs) to subtoxic concentrations of β-amyloid (0.5 μM βA) plus okadaic acid (1 nM OA), for 4 days. This treatment increased by 95 % cell death, which was mainly apoptotic as shown by positive TUNEL staining. In addition, the combination of βA/OA increased Thioflavin S aggregates, hyperphosphorylation of Tau, oxidative stress (increased DCFDA fluorescence), and neuroinflammation (increased IL-1β and TNFα). Under these experimental conditions, melatonin (1-1000 nM) and galantamine (10-1000 nM), co-incubated with the toxic stimuli, caused a concentration-dependent neuroprotection; maximal neuroprotective effect was achieved at 1 μM of melatonin and galantamine. Most effective was the finding that combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the βA/OA model. Therefore, we suggest that supplementation of melatonin in combination with lower doses of AChEIs could be an interesting strategy for AD patients.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Antioxidants; Cell Death; Galantamine; Hippocampus; Melatonin; Models, Biological; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Protein Aggregates; Rats, Sprague-Dawley; tau Proteins; Tissue Culture Techniques

Alzheimer's disease (AD) is the leading neurodegenerative disorder affecting the world's elderly population. Most experimental models of AD are transgenic or pharmacological in nature, and do not simulate the entire pathophysiology. In the present study, we developed a pharmacologically induced AD using the zebrafish, a species that can recapitulate most of the phenotypes of the disease. The pharmacological agent being used, okadaic acid (OKA) has also been utilized to study AD in other species. In this model, the immunohistochemistry of phosphorylated glycogen synthase-3α/β, Aβ, p-tau, tau protein, and senile plaque formation in zebrafish brain were all significantly increased with increasing exposure to OKA. These represent the majority of the histological hallmarks of AD pathophysiology. The observed changes were also accompanied by learning and memory deficits which are also important components in AD pathophysiology. Zebrafish disease models are gaining popularity mostly due to their economic cost and relevance to human disease pathophysiology. Current pharmacological methods of inducing AD in zebrafish are not adequately developed and do not represent all the features of the disease. OKA-induced AD in zebrafish can become a cost efficient model to study drug discovery for AD. It may also be used to unravel the molecular mechanisms underlying the complex pathophysiology that leads to AD using relatively economical species.

Topics: Alzheimer Disease; Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Maze Learning; Memory; Okadaic Acid; Zebrafish

Several lines of evidence have revealed that phosphorylation of amyloid precursor protein (APP) at Thr668 is involved in the pathogenesis of Alzheimer's disease (AD). Okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used in AD research models to increase tau phosphorylation and induce neuronal death. We previously showed that OA increased levels of APP and induced accumulation of APP in axonal swellings. In this study, we found that in OA-treated neurons, phosphorylation of APP at Thr668 increased and accumulated in axonal swellings by c-jun N-terminal kinase (JNK), and not by Cdk5 or ERK/MAPK. These results suggest that JNK may be one of therapeutic targets for the treatment of AD. [BMB Reports 2016; 49(7): 376-381].

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anthracenes; Blotting, Western; Cells, Cultured; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Neurons; Okadaic Acid; Phosphorylation; Rats; Up-Regulation

In the present study, we evaluated and compared effect of intracerebroventricular (ICV) and intrahippocampal bilateral microinjection of okadaic acid (OA) on spatial memory function assessed in one day water maze paradigm and hippocampal structure in rats. Rats were divided in following groups: Control(icv) - rats injected with ICV and aCSF; Control(hipp) - rats injected intrahippocampally with aCSF; OAicv - rats injected with ICV and OA; OAhipp - rats injected intrahippocampally with OA. Nissl staining of hippocampal sections showed that the pyramidal cell loss in OAhipp group is significantly higher than that in the OAicv. The results of behavioral experiments showed that ICV or intrahippocampal bilateral microinjection of OA did not affect learning process and short-term spatial memory but induced impairment in spatial long-term memory assessed in probe test performance 24 h after training. OA-induced spatial memory impairment may be attributed to the hippocampal cell death. Based on these results OA induced memory deficit and hippocampal cell loss in rat may be considered as a potential animal model for preclinical evaluation of antidementic drug activity.

Topics: Alzheimer Disease; Animals; Cell Count; Disease Models, Animal; Hippocampus; Male; Maze Learning; Microinjections; Okadaic Acid; Pyramidal Cells; Rats; Spatial Memory

We have investigated the protective effects of ITH91/IQM157, a hybrid of melatonin and N,N-dibenzyl(N-methyl)amine, in an in vitro model of Alzheimer's disease (AD)-like pathology that combines amyloid beta (Aβ) and tau hyperphosphorylation induced by okadaic acid (OA), in the human neuroblastoma cell line SH-SY5Y. Combination of subtoxic concentrations of Aβ and OA caused a significant toxicity of 40% cell death, which mainly was apoptotic; this effect was accompanied by retraction of the cells' prolongations and accumulation of thioflavin-S stained protein aggregates. In this toxicity model, ITH91/IQM157 (1-1000 nM) reduced cell death measured as MTT reduction; at 100 nM, it prevented apoptosis, retraction of prolongations, and Aβ aggregates. The protective actions of ITH91/IQM157 were blocked by mecamylamine, luzindol, chelerythrine, PD98059, LY294002, and SnPP. We show that the combination of melatonin with a fragment endowed with AChE inhibition in a unique chemical structure, ITH91/IQM157, can reduce neuronal cell death induced by Aβ and OA by a signaling pathway that implicates both nicotinic and melatonin receptors, PKC, Akt, ERK1/2, and induction of hemoxygenase-1.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Death; Cell Line, Tumor; Cholinesterase Inhibitors; Cytoskeleton; Heme Oxygenase-1; Humans; MAP Kinase Signaling System; Melatonin; Methylamines; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Protein Aggregates; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptors, Melatonin; Receptors, Nicotinic; tau Proteins

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

Statins are a class of cholesterol-lowering drugs and have been suggested therapeutic use for neurodegenerative diseases including Alzheimer's disease (AD). Our recent studies revealed a neuronal protective effect of lovastatin (LOV) from N-methyl-d-aspartic acid (NMDA) excitotoxicity. The neuroprotective mechanism of statins, however, is far unknown. Here we demonstrated that LOV suppressed the aberrant tau phosphorylation both from frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) mutation and okadaic acid (OA) induction in cultured rat primary neurons. The protective effect of LOV occurred at multiple pathological sites of tau protein, including Tyr181, Tyr231 Ser202/Tyr205, Tyr212/Ser214 and Ser396/Ser404. Further analysis revealed that the potential mechanism of the suppressive effect of LOV resulted from two aspects, activating OA-inhibited protein phosphatase 2A (PP2A) activity and attenuating OA-induced activity of tau kinases CDK5/P25 and CDK2/4, but not glycogen synthase kinase 3β (GSK3β). These findings give new insights into the molecular mechanism of LOV-mediated neuroprotective effect and provide experimental evidence for its therapeutic use in AD.

Topics: Alzheimer Disease; Animals; Cells, Cultured; Frontotemporal Dementia; Lovastatin; Mutation; Neurons; Okadaic Acid; Phosphorylation; Rats, Sprague-Dawley; tau Proteins

To discuss whether PP-2A influences the phosphorylation level at APP threonine 668 locus thus regulating Aβ secretion.. In the experiment, 24 hours after N2a cells of stably transfected human APP (N2a/APP) were treated with okadaic acid (OA) or DES (C6-ceramide) (N2a/APP), an injection of OA cerebral stereotactic was administered to a SD rat in the hippocampal region, or PP-2A overexpressed plasmids was transfected transiently, the aggregate levels of APP phosphorylated APP, and APP-CTF were detected through immunoblotting and the activity of PP-2A and secretase was also detected using a reagent kit.. The phosphorylation level of APP was significantly increased after the PP-2A activity was inhibited by OA. DES activated PP-2A or over-expressed PP-2A was able to reduce the phosphorylation level of APP. Either can inhibit PP and reduce the phosphorylation level of APP. The level of phosphorylated APP was increased significantly after the SD rat was injected with OA through the hippocampal region. The activity of β- and γ-secretases in N2a/APP cells significantly increased after OA treatment whereas the α-secretase activity had no significant changes; the Aβ level increased.. We discovered that PP-2A was capable of regulating the Aβ level by regulating APP phosphorylation level and β and γ-secretase activity(Fig. 5, Ref. 30).

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Ceramides; Hippocampus; Humans; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley

To study the effect of flavonoids isolated from aerial parts of Scutellaria baicalensis Georgi (SSF) on cerebral damage induced by okadaic acid (OA) in rats.. OA was microinjected into the right lateral ventricle of male rats at a dose of 200 ng kg(-1) twice with a 3-day interval between injections to establish a model of Alzheimer's-disease-like cerebral damage. Neuronal morphology was observed with thionin staining and the expressions of glial fibrillary acidic protein (GFAP) and β-amyloid peptide 1-40 (Aβ1-40) were monitored via immunohistochemistry. The level of malondialdehyde (MDA) and the activities of glutathione peroxidase (GSH-Px) and lactate dehydrogenase (LDH) were measured using spectrophotometry.. The results showed that OA-treated rats exhibited marked neuronal damage accompanied by increased levels of Aβ1-40 peptide and MDA accumulation, decreased GFAP protein expression and reduced GSH-Px and LDH activity in the brain. SSF at three doses (25, 50 and 100 mg kg(-1)) dramatically reversed the OA-induced changes in the brains of rats.. SSF-mediated amelioration of OA-induced neuronal damage in rats provides a rationale for assessing SSF as a means of to reducing tau hyperphosphorylation and Aβ expression in the treatment of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain Injuries; Disease Models, Animal; Flavonoids; Glial Fibrillary Acidic Protein; Glutathione Peroxidase; Immunohistochemistry; Injections, Intraventricular; L-Lactate Dehydrogenase; Lateral Ventricles; Male; Malondialdehyde; Microinjections; Neurons; Okadaic Acid; Oxidative Stress; Peptide Fragments; Random Allocation; Rats; Rats, Sprague-Dawley; Scutellaria baicalensis

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Topics: Aging; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Humans; Kainic Acid; Mice; Mice, Inbred C57BL; Neurons; Okadaic Acid; Peptide Fragments; Plaque, Amyloid

Green tea polyphenols (GTPs) are now being considered possible protective agents in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies suggested that GTPs could inhibit amyloid fibril formation and protect neurons from toxicity induced by β-amyloid. However, whether GTPs can ameliorate learning and memory impairments and also reduce tau hyperphosphorylation induced by okadaic acid (OA) in rats remains unclear. The aim of this study was to determine if GTPs have neuroprotection against OA-induced neurotoxicity.. In this work, rats were pretreated with GTPs by intragastric administration for 4 wk. Then OA was microinjected into the right dorsal hippocampus. Morris water maze tests were used to test the ethologic changes in all groups, and tau protein hyperphosphorylation was detected both in vivo and in vitro.. The ethologic test indicated that the staying time and swimming distance in the target quadrant were significantly decreased after OA treatment, whereas rats pretreated with GTPs stayed longer in the target quadrant. Methyl thiazolyl tetrazolium assay and lactate dehydrogenase leakage showed that GTPs greatly ameliorated primary hippocampal neurons damage induced by OA. Furthermore, reduced hyperphosphorylated tau protein was detected with GTPs pretreatment.. Taken together, our results suggest that GTPs have neuroprotection against OA-induced neurotoxicity.

Topics: Alzheimer Disease; Animals; Antioxidants; Hippocampus; Learning; Maze Learning; Memory Disorders; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Polyphenols; Rats; Rats, Sprague-Dawley; tau Proteins; Tea

The activity of protein phosptase-2A (PP2A) is significantly decreased in the brains of Alzheimer's disease (AD) patients, but the upstream effectors for regulating PP2A activity are not fully understood. Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) is a key enzyme involved in energy metabolism and its gene expression level is reduced in AD brain specimens. Whether Nmnat2 can activate PP2A deserves to be explored. Here, we first measured the level of Nmnat2, Tyr307-phosphorylation of PP2A, and tau phosphorylation in Tg2576 mice. We observed that the mRNA and protein levels of Nmnat2 were significantly decreased with a simultaneous elevation of p-Tyr307-PP2A and tau phosphorylation in Tg2576 mice. Further studies in HEK293 cells with stable expression of human tau441 (HEK293/tau) demonstrated that simultaneous inhibition of PP2A by okadaic acid abolished the Nmnat2-induced tau dephosphorylation. Moreover, we further demonstrated that overexpression of Nmnat2 could activate PP2A with attenuation of tau phosphorylation, whereas downregulation of Nmnat2 by shRNA inhibited PP2A with tau hyperphosphorylation at multiple AD-associated sites. Our data provide the first evidence that Nmnat2 affects tau phosphorylation by regulating PP2A activity, suggesting that Nmnat2 may serve as a potential target in arresting AD-like tau pathologies.

Topics: Alzheimer Disease; Animals; Blotting, Western; Disease Models, Animal; Down-Regulation; Enzyme Activation; HEK293 Cells; Humans; Mice; Nicotinamide-Nucleotide Adenylyltransferase; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Real-Time Polymerase Chain Reaction; tau Proteins

Ginsenoside Rg1, one of the major active ingredients isolated from Panax Ginseng, has been shown notable neuroprotective effects in memory impairment animals. However, the role of ginsenoside Rg1 on cognition capacity damaged by neurofibrillary tangles (NFTs) is still poorly understood, and the underlying mechanism remain to be fully elucidated. Okadaic acid (OKA), a potent phosphatase inhibitor, often apply to imitate Alzheimer's disease-like symptom damaged by neurofibrillary tangles, was used to investigate the effects of ginsenoside Rg1 on memory impairment and the related mechanisms in Sprague Dawley (SD) rats. The anti-dementic drug donepezil was used as a positive contrast. The results showed that OKA intracerebroventricular (i.c.v.) injection induced memory impairment, including changes in the ability of orientation navigate, spatial probe and relearning memory in behavioral test of Morris water maze (MWM). However, treatment with Rg1 and donepezil remarkably alleviated these changes. Also OKA treated rats showed memory impairment including increasing of phospho-tau, decreasing of phospho-GSK3β and the formation of β-amyloid in special brain regions, which were reversed by Rg1 (20 mg/kg) and donepezil (1 mg/kg) administration. All these indicating that ginsenoside Rg1 protects rats against OKA-induced neurotoxicity. The possible neuroprotective mechanism may be that Rg1 decreases OKA-induced memory impairment through GSK3β/tau signaling pathway and/or attenuating Aβ formation. Thus, these studies indicate that ginsenoside Rg1 might be a potential preventive drug for Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Ginsenosides; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Male; Maze Learning; Memory Disorders; Neuroprotective Agents; Okadaic Acid; Parietal Lobe; Rats; Rats, Sprague-Dawley; tau Proteins

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

Upregulation of the lysosomal system has been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). But the exact role of this system remains unknown. Okadaic acid (OA), a protein phosphatase-2A inhibitor, increases tau phosphorylation, β-amyloid deposition, and neuronal cell death, which are the pathological hallmarks of AD. To investigate the role of lysosomal activation in AD brain cells, cultured neurons were treated with OA and assessed lysosomal morphology and enzyme activity and the protective effect of cathepsin B, D, or L inhibitors. It was found that although it induced lysosomal swelling and enzyme activation, OA did not induce lysosomal rupture. While inhibition of cathepsin D and L failed to protect neurons from OA-induced cell death, CA074-Me, a cathepsin B inhibitor, conferred a protective effect. Interestingly, CA-074Me reduced amyloid precursor protein (APP) accumulation and α-spectrin cleavage, similar to the effect of calpain inhibition.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Cathepsin B; Cells, Cultured; Dipeptides; Down-Regulation; Neocortex; Neurons; Neuroprotective Agents; Okadaic Acid; Rats; Treatment Outcome

To investigate the effect of acteoside on SK-N-SH nerve cell injury induced by okadaic acid (OA).. SK-N-SH nerve cells were processed with 20 nmol * L OA to establish the Alzheimer's disease (AD) cellular model, and 5, 10, 20 mg . L-1 acteoside was used to antagonize against its effect. Cell morphology was observed under inverted microscope. The cell survival rate was detected with MTT, and the LDH release rate was measured by enzyme label kit. Western blot was applied to determine the expression of phosphorylation tau proteins in nerve cells.. The acteoside could significantly improve SK-N-SH cell morphology, enhance the cell survival rate, decrease the cell LDH release rate and the expression of phosphorylated tau proteins at p-Ser 199/202 and p-Ser 404 sites, up-regulated the expression of at non-phosphorylated tau proteins at Ser 202 site and Ser 404 sites.. Acteoside has significant protective effect on nerve cell injury induced by OA.

Topics: Alzheimer Disease; Cell Line; Cell Survival; Glucosides; Humans; Okadaic Acid; Phenols; tau Proteins

Failures in neurotrophic support and signalling play key roles in Alzheimer's disease (AD) pathogenesis. We previously demonstrated that downregulation of the neurotrophin effector Kinase D interacting substrate (Kidins220) by excitotoxicity and cerebral ischaemia contributed to neuronal death. This downregulation, triggered through overactivation of N-methyl-D-aspartate receptors (NMDARs), involved proteolysis of Kidins220 by calpain and transcriptional inhibition. As excitotoxicity is at the basis of AD aetiology, we hypothesized that Kidins220 might also be downregulated in this disease. Unexpectedly, Kidins220 is augmented in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlates with enhanced Kidins220 resistance to calpain processing but no higher gene transcription. Using AD brain necropsies, glycogen synthase kinase 3-β (GSK3β)-transgenic mice and cell models of AD-related neurodegeneration, we show that GSK3β phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. As altered activities of GSK3β and phosphatases are involved in tau aggregation and constitute hallmarks in AD, a GSK3β/PP1 imbalance may also contribute to Kidins220 decreased clearance, accumulation and hampered neurotrophin signalling from early stages of the disease pathogenesis. These results encourage searches for mutations in Kidins220 gene and their possible associations to dementias. Finally, our data support a model where the effects of excitotoxicity drastically differ when occurring in cerebral ischaemia versus progressively sustained toxicity along AD progression. The striking differences in Kidins220 stability resulting from chronic versus acute brain damage may also have important implications for the therapeutic intervention of neurodegenerative disorders.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Brain Ischemia; Calpain; Cell Death; Cells, Cultured; Disease Models, Animal; Down-Regulation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Male; Membrane Proteins; Mice; Mice, Transgenic; Nerve Growth Factors; Nerve Tissue Proteins; Neurodegenerative Diseases; Neurons; Okadaic Acid; Phosphorylation; Protein Phosphatase 1; Proteolysis; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Signal Transduction; tau Proteins

Cerebral okadaic acid (OA) administration induces Alzheimer's disease (AD)-like phenotype in rats. Alterations in glutamate levels associated with hyperactivation of cyclin dependent kinase 5 (Cdk5) signaling pathway downstream Tau phosphorylation may participate in the genesis of this pathological phenotype. Here, we examined the efficacy of memantine (MN) pretreatment on reducing OA-induced AD-like phenotypes in rats. Wistar rats were given daily intraperitoneal injections of MN for 3 days and then given an intrahippocampal infusion of OA. Animals were divided into four groups: control (CO), MN, OA and MN/OA. Spontaneous locomotion and spatial memory performance were assessed by open field and Morris water maze respectively. Additionally, we measured glutamate levels in the cerebrospinal fluid (CSF) and the immunocontent of Cdk5, p35, p25 and phosphorylated Tau (pTauSer199/202) in the hippocampus. Spontaneous locomotion did not differ between groups. The OA group showed a significant decrease in spatial memory performance compared to all groups. The OA infusion also increased CSF glutamate levels and the immunocontents of Cdk5, p25 and pTauSer199/202 in the hippocampus. Conversely, pretreatment with MN prevented OA-induced spatial memory deficits and the increment of CSF glutamate level; which paralleled with normal immunocontents of Cdk5, p25 and pTau- Ser199/202 proteins. There were positive correlations between spatial memory performance and the neurochemical parameters. In summary, pretreatment with MN prevents spatial memory deficits induced by intrahippocampal OA administration in rats. The prevention of increase CSF glutamate levels, along with the reduced hippocampal phosphorylation of TauSer199/202 by Cdk5/p25 signaling pathway, are the mechanisms proposed to participate in the prophylactic effects of MN in this AD-like model.

Topics: Alzheimer Disease; Animals; Carcinogens; Chromatography, High Pressure Liquid; Cyclin-Dependent Kinase 5; Disease Models, Animal; Drug Administration Schedule; Excitatory Amino Acid Antagonists; Exploratory Behavior; Glutamic Acid; Hippocampus; Locomotion; Male; Maze Learning; Memantine; Okadaic Acid; Rats; Rats, Wistar; Signal Transduction; Statistics as Topic; tau Proteins

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

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

We have previously reported the multifunctional profile of N-(3-chloro-10H-phenothiazin-10-yl)-3-(dimethylamino)propanamide (1) as an effective neuroprotectant and selective butyrylcholinesterase inhibitor. In this paper, we have developed a series of N-acylaminophenothiazines obtained from our compound library or newly synthesised. At micro- and sub-micromolar concentrations, these compounds selectively inhibited butyrylcholinesterase (BuChE), protected neurons against damage caused by both exogenous and mitochondrial free radicals, showed low toxicity, and could penetrate into the CNS. In addition, N-(3-chloro-10H-phenothiazin-10-yl)-2-(pyrrolidin-1-yl)acetamide (11) modulated the cytosolic calcium concentration and protected human neuroblastoma cells against several toxics, such as calcium overload induced by an L-type Ca2+-channel agonist, tau-hyperphosphorylation induced by okadaic acid and Aβ peptide.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Antineoplastic Agents; Butyrylcholinesterase; Calcium; Cell Death; Cell Survival; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Humans; Molecular Structure; Okadaic Acid; Peptide Fragments; Phenothiazines; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

Panax ginseng, a traditional Chinese herbal medicine, has been widely used to restore the disease and enhance the healthy body in Asia for about 5000 years. The present study aimed to investigate the possible neuroprotective effects of ginsenoside Rd against OA-induced toxicity.. Ginsenoside Rd was used in tauopahy models of Alzheimer's disease (AD). To mimic the in vivo or in vitro tau hyperphosphorylation, okadaic acid (OA), a protein phosphatase inhibitor, was bilaterally micro-infused into the cerebral ventricle of adult male Sprague-Dawley (SD) rats, or added in media of cultured cortical neurons. The phosphorylation levels of tau and the activities of protein phosphatase 2A (PP-2A) were measured and compared with ginsenoside Rd pretreated groups.. Pretreatment with ginsenoside Rd in SD rats (10mg/kg for 7 days) or in cultured cortical neurons (2.5 or 5μmol/L for 12h) reduced OA-induced neurotoxicity and tau hyperphosphorylation by enhancing the activities of PP-2A.. The result of the present work implied that ginsenoside Rd protected SD rats and cultured cortical neurons against OA-induced toxicity. The possible neuroprotective mechanism may be that ginsenoside Rd decreases OA-induced the hyperphosphorylation of tau by the increase in activities of PP-2A. Thus, this study promises that ginsenoside Rd might be a potential preventive drug candidate for AD and other tau pathology-related neuronal degenerative diseases.

Topics: Alzheimer Disease; Animals; Brain; Cells, Cultured; Disease Models, Animal; Ginsenosides; Male; Neuroprotective Agents; Okadaic Acid; Panax; Phosphorylation; Phytotherapy; Plant Extracts; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; tau Proteins

1,8-Naphthyridine derivatives related to 17 (ITH4012), a neuroprotective compound reported by our research group, have been synthesized. In general, they have shown better inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) than most tacrine derivatives previously synthesized in our laboratory. The compounds presented an interesting neuroprotective profile in SH-SY5Y neuroblastoma cells stressed with rotenone/oligomycin A. Moreover, compound 14 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) also caused protection in cells stressed with okadaic acid (OA) or amyloid beta 1-42 peptide (Abeta(1-42)). Interestingly, compound 14 prevented the OA-induced PP2A inhibition, one of the enzymes implicated in tau dephosphorylation. This compound also exhibited neuroprotection against neurotoxicity elicited by oxygen and glucose deprivation in hippocampal slices. Because these stressors caused neuronal damage related to physiopathological hallmarks found in the brain of Alzheimer's disease (AD) patients, we conclude that compound 14 deserves further in vivo studies in AD models to test its therapeutic potential in this disease.

Topics: Acetylcholinesterase; Alzheimer Disease; Amyloid beta-Peptides; Animals; Butyrylcholinesterase; Calcium; Catalytic Domain; Cell Death; Cell Line, Tumor; Cholinesterase Inhibitors; Cytotoxins; Electron Transport Chain Complex Proteins; Electrophorus; Glucose; Hippocampus; Humans; Isomerism; Models, Molecular; Naphthyridines; Neuroprotective Agents; Okadaic Acid; Oligomycins; Oxygen; Peptide Fragments; Protein Binding; Protein Phosphatase 2; Rats; Rotenone; Structure-Activity Relationship

Glycogen synthase kinase-3beta (GSK3beta) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3beta. However, the inactive form of GSK3beta which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3beta substrates, such as beta-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3beta at serine-9 and other substrates including tau, beta-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3beta inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3beta may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3beta inhibitors could be a valuable drug candidate in AD.

Topics: Alzheimer Disease; Animals; Cells, Cultured; Disease Models, Animal; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Indoles; Lithium Chloride; Neurons; Okadaic Acid; Oximes; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Rats; Serine

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes cognitive and behavioral deterioration in the elderly. Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of AD that has been shown to correlate positively with the severity of dementia in the neocortex of AD patients. In an attempt to characterize an in vivo AD tauopathy model, okadaic acid (OA), a protein phosphatase inhibitor, was microinfused into the right lateral dorsal hippocampus area of ovariectomized adult rat. Cognitive deficiency was seen in OA-treated rats without a change in motor function. Both silver staining and immunohistochemistry staining revealed that OA treatment induces NFTs-like conformational changes in both the cortex and hippocampus. Phosphorylated tau as well as cyclin-dependent kinase 5 (cdk5) and its coactivator, p25, were significantly increased in these regions of the brain. Oxidative stress was also increased with OA treatment as measured by protein carbonylation and lipid peroxidation. These data suggest that the unilateral microinfusion of OA into the dorsal hippocampus causes cognitive deficiency, NFTs-like pathological changes, and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases.

Topics: Alzheimer Disease; Animals; Blotting, Western; Brain; Cognition Disorders; Disease Models, Animal; Enzyme Inhibitors; Female; Immunohistochemistry; Injections, Intraventricular; Maze Learning; Neurofibrillary Tangles; Okadaic Acid; Ovariectomy; Oxidative Stress; Phosphorylation; Rats; Rats, Sprague-Dawley; Tauopathies

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Topics: Adenylate Kinase; Alzheimer Disease; Animals; Cells, Cultured; Enzyme Inhibitors; Epitopes; HeLa Cells; Humans; Hypoglycemic Agents; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Transgenic; Multiprotein Complexes; Neurofibrillary Tangles; Neurons; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Proteins; Signal Transduction; tau Proteins; TOR Serine-Threonine Kinases

Transforming growth factors beta (TGFbeta) regulate multiple biological activities. TGFbeta activation of the Smad pathway results in activation of genes encoding extracellular matrix molecules, proteases, protease activators and protease inhibitors. In Alzheimer's disease (AD), TGFbeta protein and mRNA levels are raised, which would be expected to be neuroprotective. However, recent observations suggest that TGFbeta-Smad signalling is disrupted by the hyperphosphorylation of tau, the primary component of neurofibrillary tangles: phosphorylated Smad2/3 (pSmad 2/3) co-localises with phosphorylated tau in the neuronal cytoplasm and levels are reduced in the nucleus. We have investigated whether in vitro induction of tau hyperphosphorylation influences pSmad 2/3 localisation in rat primary cortical cells. Treatment with okadaic acid, a protein phosphatase 1 and 2A inhibitor caused hyperphosphorylation of tau at epitopes hyperphosphorylated in AD and disrupted pSmad 2/3 translocation into the nucleus. The disruptive effect of tau phosphorylation on pSmad 2/3 translocation was confirmed by treatment of primary cortical cells with synthetic oligomeric A beta(1-42), a more physiologically relevant model of AD. Our findings suggest that despite the increased level of TGFbeta in AD, the TGFbeta-Smad signalling pathway is impeded within neurones due to sequestration of pSmad 2/3 by hyperphosphorylated tau. This may compromise neuroprotective actions of TGFbeta and contribute to neurodegeneration in AD.

Topics: Active Transport, Cell Nucleus; Alzheimer Disease; Amyloid beta-Peptides; Animals; Blotting, Western; Cell Nucleus; Cells, Cultured; Cerebral Cortex; Cytoplasm; Enzyme Inhibitors; Fluorescent Antibody Technique; Immunoprecipitation; Okadaic Acid; Peptide Fragments; Phosphorylation; Rats; Smad2 Protein; Smad3 Protein; tau Proteins

We investigated whether neural stem cells (NSC) with transgenic expression of human nerve growth factor (hNGF) transplanted into the brain could offer a therapeutic option for the treatment of Alzheimer's disease (AD).. We infused okadaic acid into rat lateral ventricles to establish a chronic AD animal model. In addition, NSC were stably transduced with hNGF and enhanced green fluorescent protein (eGFP) genes (NSC-hNGF-eGFP) by using a recombination adeno-associated virus serotype 2 (rAAV2) vector. These genetically modified stem cells were grafted into the cerebral cortex of AD rats.. AD model rats showed significant damage in learning and memory function, with the formation of senile plaques and neurofibrillary tangles in the cerebral cortex. The transferred hNGF gene conferred stable and high levels of protein expression in NSC in vitro. Moreover, the NSC-hNGF-eGFP, but not the NSC, survived, integrating into the host brain and enhancing cognitive performance after transplantation.. The injection of okadaic acid into rat lateral ventricles constitutes a promising animal model for investigating selective aspects of AD. rAAV2-mediated hNGF delivery can render long-term and stable transduction of hNGF in NSC. NSC-hNGF-eGFP transplantation may offer a viable therapeutic approach for treatment of AD.

Topics: Alzheimer Disease; Animals; Dependovirus; Disease Models, Animal; Fetus; Genetic Vectors; Humans; Learning; Male; Nerve Growth Factor; Neurons; Okadaic Acid; Rats; Recombinant Proteins; Stem Cell Transplantation; Stem Cells; Transduction, Genetic

Autophagosomes are accumulated in Alzheimer's disease (AD), but the regulatory pathway of autophagy in AD remains largely unknown. By using electron microscopy, Western blotting, and immunocytochemistry, here we show that autophagosomes are accumulated in rat neurons by okadaic acid (OA), a protein phosphatase-2A inhibitor known to enhance tau phosphorylation, beta-amyloid (Abeta) deposition, and neuronal death, which are the pathological hallmarks of AD. Autophagy can be generally induced via several distinct pathways, such as inhibition of mTOR or activation of beclin-1. Interestingly, OA increased both mTOR and beclin-1 pathways simultaneously, which suggests that autophagy in OA-treated neurons is induced mainly via the beclin-1 pathway, and less so via mTOR inhibition. Finally, inhibition of autophagy by 3MA reduced cytotoxicity in OA-treated neurons. Our novel findings provide new insights into the pathology of and therapeutic intervention for AD.

Topics: Adenine; Alzheimer Disease; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Enzyme Inhibitors; Immunohistochemistry; Lysosomes; Microscopy, Electron, Transmission; Neurons; Neuroprotective Agents; Okadaic Acid; Phagosomes; Protein Kinases; Rats; TOR Serine-Threonine Kinases

Senile plaques and neurofibrillary tangles (NFTs) represent two of the major histopathological hallmarks of Alzheimer's disease (AD). The plaques are primarily composed of aggregated amyloid beta (Abeta) peptides. The processing of amyloid-beta precursor protein (AbetaPP) in okadaic acid (OA)-induced tau phosphorylation primary neurons was studied.. Primary cultures of rat brain cortical neurons were treated with OA and beta-secretase inhibitor. Neurons' viability was measured. AbetaPP processing was examined by immunocytochemistry and Western blotting with specific antibodies against the AbetaPP-N-terminus (NT) and AbetaPP-C-terminus (CT).. Ten nmol/L OA had a time-dependent suppression effect on primary neurons' viability. The suppression effect was alleviated markedly by pretreatment with beta-secretase inhibitor. After OA treatment, both AbetaPP and beta-C-terminal fragment (betaCTF) were significantly increased in neurons. AbetaPP level was increased further in neurons pretreated with beta-secretase inhibitor.. In OA-induced tau phosphorylation cell model, inhibition of beta-secretase may protect neurons from death induced by OA. Because of increased accumulation of AbetaPP in neurons after OA treatment, more AbetaPP turns to be cleaved by beta-secretase, producing neurotoxic betaCTF. As apotential effective therapeutic target, beta-secretase is worth investigating further.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Blotting, Western; Cell Survival; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Immunohistochemistry; Okadaic Acid; Peptide Fragments; Rats

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

We have investigated using single channel patch-clamp methods potassium channel prevalence in hippocampal neurones from two animal models of AD. Experiments have been carried out on transgenic mice (Tg2576) carrying the Swedish mutation (K670N/M671L) and rats receiving ventricular infusions of okadaic acid. In cell-attached patches from hippocampal neurones from the Tg2576 and control littermate mice there were three principal unitary conductance - 22 pS, 111 pS and 178 pS. The two channels of intermediate and large conductance were voltage-dependent, highly active in cell-attached patches, activity decreasing markedly on hyperpolarisation. The large conductance channel was sensitive to TEA, iberiotoxin, was activated in excised inside-out patches by Ca 2+(i) and is the type I maxi-K+ channel. Significantly, there was a reduction in the prevalence of a TEA-sensitive 113 pS channel in neurones from TG2576 mice with a corresponding increase in prevalence of the maxi-K+ channel. There was no difference in the characteristics of maxi-K+ between patches in neurones from the transgenic and littermate controls. In the rat model single channel analysis was performed on hippocampal neurons from three groups of animals i.e. non-operated, and these receiving an infusion of vehicle or vehicle with okadaic acid. Three principal unitary conductances of around 18 pS, 118 pS and 185 pS were also observed in cell-attached recordings from these three groups. The intermediate and high conductance channels were blocked by TEA or 4-AP or 140 mM RbCl. There were no statistically significant differences in the channel prevalence or channel density between the control and test groups.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Disease Models, Animal; Enzyme Inhibitors; Hippocampus; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neurons; Okadaic Acid; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Inbred Strains; Rats, Sprague-Dawley

In order to understand how plasticity is related to neurodegeneration, we studied synaptic proteins with quantitative immunohistochemistry in the entorhinal cortex from Alzheimer patients and age-matched controls. We observed a significant decrease in presynaptic synaptophysin and an increase in postsynaptic density protein PSD-95, positively correlated with beta amyloid and phosphorylated Tau proteins in Alzheimer cases. Furthermore, Alzheimer-like neuritic retraction was generated in okadaic acid (OA) treated SH-SY5Y neuroblastoma cells with no decrease in PSD-95 expression. However, in a SH-SY5Y clone with decreased expression of transcription regulator LMO4 (as observed in Alzheimer's disease) and increased neuritic length, PSD-95 expression was enhanced but did not change with OA treatment. Therefore, increased PSD-95 immunoreactivity in the entorhinal cortex might result from compensatory mechanisms, as in the SH-SY5Y clone, whereas increased Alzheimer-like Tau phosphorylation is not related to PSD-95 expression, as suggested by the OA-treated cell models.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Cell Line, Tumor; Cell Survival; Disks Large Homolog 4 Protein; Female; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Neurites; Okadaic Acid; Phosphorylation; tau Proteins; Up-Regulation

Impairment of protein phosphatase 2A (PP2A) activity is implicated in tau hyperphosphorylation and microtubule (MT) instability in Alzheimer's disease (AD). Here, we report that okadaic acid, an effective PP2A inhibitor, suppresses the levels of acetylated and detyrosinated tubulins, but enhances tyrosinated tubulins in rat primary cortical neuron cultures. Immunocytochemistry experiments reveal that MTs accumulate intensely around soma and proximal neurites, implying impairment of MT transport to distal neurites which is mediated by dynein and dynactin. Here, we reveal that they can be cleaved by calpain. Notably, shortening of process length in OA-treated neurons is alleviated when calpain cleavage activity is inhibited. Based on these results, we propose that calpain-mediated dynein cleavage in OA-treated neurons is responsible for the MT transport deficit, and consequently, neurite retraction.

Topics: Acetylation; Alzheimer Disease; Animals; Biological Transport; Calcium Chloride; Calcium Hydroxide; Cells, Cultured; Cerebral Cortex; Drug Combinations; Dyneins; Enzyme Inhibitors; Microtubules; Neurons; Okadaic Acid; Phosphorylation; Potassium Chloride; Protein Phosphatase 2; Protein Processing, Post-Translational; Rats; Sodium Bicarbonate; Sodium Chloride; Tubulin; Tyrosine

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

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

The present study was aimed to investigate the effects of ginsenoside Rb1 on okadaic acid (OA)-induced Tau hyperphosphorylation in hippocampal neurons of Sparague-Dawley rat and to explore its possible mechanism. Animals were randomly divided into four groups. Group 1 received dimethysulphoxide (DMSO) injection (vehicle group), group 2 only received OA injection (OA group), group 3 was pretreated with Rb1 and then received OA injection (Rb1 pretreatment group), and the group 4 was an intact control group. The animals in group 3 were injected intraperitoneally with various doses of Rb1 at 5, 10, and 20 mg/kg (once a day for 14 d). On the thirteen day of pretreatment, animals in Rb1 pretreatment group as well as animals in OA group received a bolus injection of 0.483 microg of OA (1.5 microl of solution in DMSO) at right dorsal aspect of hippocampus to induce Tau hyperphosphrylation. The brains were harvested one day after the last treatment. In all groups, the morphology of neurofibrils, phosphorylation of Tau protein, and the activity of phosphatase 2A (PP2A) were investigated. In OA group, the Bielschowski's assay revealed darkened and uneven neurofibrils staining in the hippocampus. The immunohistochemistry results showed a significant increase in Thr(231) phosphorylation of Tau protein in OA group relative to the control group (P<0.01). OA injection also markedly decreased PP2A activity (P<0.01). Western blot confirmed Thr(231) phosphorylation of Tau protein and it also detected phosphorylation of Ser(396) of Tau protein. The animals with Rb1 pretreatment displayed even staining of neurofibrils and normal pattern of fiber organization. Rb1 pretreatment also attenuated Thr(231) and Ser(396) hyperphosphorylations of Tau protein, and restored PP2A activity compared to the OA group (P<0.01). These results indicate that OA-induced hyperphosphorylation of Tau protein in rat hippocampal neurons can be attenuated by the pretreatment of ginsenoside Rb1. These data also implicate that Rb1 has potential neuroprotective effects on Tau-related neuropathology.

Topics: Alzheimer Disease; Animals; Ginsenosides; Hippocampus; Male; Neurons; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Random Allocation; Rats; Rats, Sprague-Dawley; tau Proteins

We isolated and characterized several phosphoseryl/phosphothreonyl phosphatase activities (P1-P11) from frontal lobe of six autopsied human brains. Of these, PP1 (P3) was a major tau phosphatase. The enzyme required metal ions and was maximally activated by Mn2+. Western blots with antibodies to known protein phosphatases showed PP1 and PP2B immunoreactivity. However, the removal of PP2B by immunoabsorption or its inhibition with EGTA did not result in appreciable loss of P3 activity. These observations suggest that P3 was an enriched PPI. Dephosphorylation of Alzheimer disease hyperphosphorylated tau (AD P-tau) by PP1 was site-specific. PPI preferentially dephosphorylated pT212 (40%), pT217 (26%), pS262 (33%), pS396 (42%) and pS422 (31%) of AD P-tau. Dephosphorylation of tau at pT181, pS199, pS202, pT205, pS214, and pS404, was undetectable. Of the sites dephosphorylated, pT212 was only a substrate for PP1, as purified/enriched PP2A and PP2B from the same brains did not dephosphorylate this site.

Topics: Adenosine Diphosphate; Alzheimer Disease; Aniline Compounds; Blotting, Western; Brain Chemistry; Calcineurin; Calmodulin; Chromatography, Agarose; Chromatography, DEAE-Cellulose; Chromatography, Gel; Enzyme Inhibitors; Humans; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Polylysine; Protein Phosphatase 1; Recombinant Proteins; Substrate Specificity; tau Proteins; Triazines

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

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

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

Neurofibrillary tangles (NFTs) are classic lesions of Alzheimer's disease. NFTs are bundles of abnormally phosphorylated tau, the paired helical filaments. The initiating mechanisms of NFTs and their role in neuronal loss are still unknown. Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in neuronal death observed in brains of patients with Alzheimer's disease. Alterations in tau phosphorylation and tau cleavage by caspases have been previously reported in neuronal apoptosis. However, the links between the alterations in tau phosphorylation and its proteolytic cleavage have not yet been documented. Here, we show that, during staurosporine-induced neuronal apoptosis, tau first undergoes transient hyperphosphorylation, which is followed by dephosphorylation and cleavage. This cleavage generated a 10-kDa fragment in addition to the 17- and 50-kDa tau fragments previously reported. Prior tau dephosphorylation by a glycogen synthase kinase-3beta inhibitor, lithium, enhanced tau cleavage and sensitized neurons to staurosporine-induced apoptosis. Caspase inhibition prevented tau cleavage without reversing changes in tau phosphorylation linked to apoptosis. Furthermore, the microtubule depolymerizing agent, colchicine, induced tau dephosphorylation and caspase-independent tau cleavage and degradation. Both phenomena were blocked by inhibiting protein phosphatase 2A (PP2A) by okadaic acid. These experiments indicate that tau dephosphorylation precedes and is required for its cleavage and degradation. We propose that the absence of cleavage and degradation of hyperphosphorylated tau (due to PP2A inhibition) may lead to its accumulation in degenerating neurons. This mechanism may contribute to the aggregation of hyperphosphorylated tau into paired helical filaments in Alzheimer's disease where reduced PP2A activity has been reported.

Topics: Alzheimer Disease; Animals; Apoptosis; Caspase Inhibitors; Cells, Cultured; Cerebral Cortex; Colchicine; Embryo, Mammalian; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Lithium; Neurons; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Wistar; Staurosporine; 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

In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.

Topics: Alzheimer Disease; Animals; Enzyme Activation; Enzyme Inhibitors; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Ribosomal Protein S6 Kinases, 70-kDa

The serine/threonine phosphatase 2A (PP2A) has been implicated in the pathogenesis of Alzheimer's disease (AD) due to its important role in regulating dephosphorylation of the microtubule-associated protein tau and mitogen-activated protein (MAP) kinase. In the present study, we show that PP2A was responsible for dephosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) following its activation by BK stimulation. Abnormal gene and protein expressions of PP2A, as well as its activity, were found to contribute to the abnormally prolonged Erk1/2 phosphorylation in the AD fibroblasts. Inhibition of PP2A with okadiac acid produced enhanced and more lasting Erk1/2 phosphorylation after BK stimulation, whereas FK506, an inhibitor of PP2B and FK-binding protein, inhibited the BK-stimulated Erk1/2 phosphorylation. Furthermore, while the phosphorylated Erk1/2 was concentrated in the nucleus of AC cells, it was mainly distributed in the extranuclear compartments of AD cells. These results suggest that the delayed dephosphorylation of Erk1/2 in AD cells following its BK-stimulated activation may be due to deficits of PP2A activity and impaired nuclear translocation of phosphorylated Erk1/2.

Topics: Active Transport, Cell Nucleus; Aged; Aged, 80 and over; Alzheimer Disease; Cell Compartmentation; Cells, Cultured; Enzyme Inhibitors; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Middle Aged; Mitogen-Activated Protein Kinases; Okadaic Acid; Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; RNA, Messenger; Tacrolimus; Tacrolimus Binding Proteins

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

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

To explore the effect of Alzheimer-like protein phosphatase deficiency on neurofilament phosphorylation.. Cell culture, light microscopy, immunocytochemistry and biochemistry techniques were used to make a phosphatase deficient cell model, to detect cell morphology, neurofilament phosphorylation and distribution, cell viability and activity.. Non-phosphorylated neurofilament recognized by SMI32 was detected both in cell body and cell processes, it was extremely enriched in cell bodies; Phosphorylated neurofilament bound to SMI34 was mainly determined in cell processes and cell surface. After treatment with okadaic acid (OA), non-phosphorylation-dependent antibodies SMI32 staining was significantly decreased in the cell body, whereas phosphorylated neurofilament reacted with SMI34 was strikingly increased in immunocytochemistry and Western blot, and prominently accumulated to the same cell location. Accompanied with hyper-phosphorylation and accumulation of neurofilament, dose dependent cell toxicity was observed by okadaic acid treatment.. Deficiency in protein phosphatase induces in neuroblastoma cell line, neurofilament phosphorylation and accumulation, which is involved in Alzheimer neurofibrillary degeneration.

Topics: Alzheimer Disease; Enzyme Inhibitors; Humans; Neurofilament Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Tumor Cells, Cultured

The reversible protein phosphorylation is the most important cellular regulation of the biological functions of many proteins. Disregulation of protein phosphorylation is involved in pathogeneses of several human diseases. The abnormal hyperphosphorylation of microtubule-associated protein tau and its aggregation into neurofibrillary tangles in selective neurons is one of the major brain pathologies of Alzheimer's disease and several other related neurodegenerative diseases. Here we present metabolically competent rat brain slices as a model to study the regulation of protein phosphorylation in brain. Employing this model we have been able to study the abnormal hyperphosphorylation of tau and other microtubule-associated proteins. We have evaluated the activity and intactness of the rat brain slices both biochemically and morphologically. Selective inhibition of protein phosphatase 2A in these rat brain slices by the treatment with okadaic acid induced hyperphosphorylation of tau at many abnormal sites seen in Alzheimer's disease brain and the accumulation of hyperphosphorylated tau in pyramidal neurons of the cortex and hippocampus. The regulation of the phosphorylation of high-molecular-weight microtubule-associated protein, MAP1b, was also studied with this model. This model enables studies on the regulation of protein phosphorylation not only biochemically, but also histochemically and immunocytochemically. Furthermore, unlike cultured cells, the neurons in the brain slices reside in the physiological environment of the brain consisting of natural extracellular matrix, neuronal connectivity, and neuronal-glial interactions.

Topics: Alzheimer Disease; Animals; Blotting, Western; Brain; Cyclosporine; Enzyme Inhibitors; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Male; Microtubule-Associated Proteins; Nuclear Proteins; Okadaic Acid; Organ Culture Techniques; Phosphorylation; Proteins; Rats; Rats, Wistar; RNA-Binding Proteins; tau Proteins

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3 beta (TPKI/GSK3 beta), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3 beta and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3 beta and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD.

Topics: Alzheimer Disease; Animals; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Food Deprivation; Glycogen Synthase Kinase 3; Hippocampus; Humans; Lithium Chloride; Male; Mice; Mice, Inbred C57BL; Models, Biological; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Proteins; Time Factors; Up-Regulation

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

Fibronectin appears to be present in Senile Plaques of Alzheimer's disease brains. These senile or neuritic plaques are surrounded by dystrophic neurites, activated microglia and reactive astrocytes. The purpose of this work was to establish if a direct correlation exists between the production of Fibronectin (FN) by astrocytes and the presence of amyloid, analysing the modification of this protein produced after the treatment of cultured astrocytes with amyloid peptide (25-35). Our data showed that the addition of previously polymerised A beta-peptide to cultured astrocytes induced a marked increase in FN immunoreactivity that is in part dependent on phosphatases 2A or phosphatase 1, since was partially inhibited by okadaic acid. The increased amount of FN did not appear to be associated to any specific single isoform of which are mainly present in the rat brain. Our data suggest that in vivo FN accumulated in senile plaques may be the result, at least in part, of the response of reactive astrocyte to the presence of amyloid peptide. The importance of FN up-regulation in vivo, as part of a 'positive' response of the astrocytes to produce molecules that favours neurite outgrowth, is discussed.

Topics: Alternative Splicing; Alzheimer Disease; Amyloid beta-Peptides; Animals; Animals, Newborn; Astrocytes; Cell Differentiation; Cell Size; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Fibronectins; Glial Fibrillary Acidic Protein; Gliosis; Growth Substances; Immunohistochemistry; Neurites; Okadaic Acid; Peptide Fragments; Plaque, Amyloid; Protein Isoforms; Rats; RNA, Messenger; Vimentin

Hyperphosphorylated tau, which is the major protein of the neurofibrillary tangles in Alzheimer's disease brain, is most probably the result of an imbalance of tau kinase and phosphatase activities in the affected neurons. By using metabolically competent rat brain slices as a model, we found that selective inhibition of protein phosphatase 2A by okadaic acid induced an Alzheimer-like hyperphosphorylation and accumulation of tau. The hyperphosphorylated tau had a reduced ability to bind to microtubules and to promote microtubule assembly in vitro. Immunocytochemical staining revealed hyperphosphorylated tau accumulation in pyramidal neurons in cornu ammonis and in neocortical neurons. The topography of these changes recalls the distribution of neurofibrillary tangles in Alzheimer's disease brain. Selective inhibition of protein phosphatase 2B with cyclosporin A did not have any significant effect on tau phosphorylation, accumulation, or function. These studies suggest that protein phosphatase 2A participates in regulation of tau phosphorylation, processing, and function in vivo. A down-regulation of protein phosphatase 2A activity can lead to Alzheimer-like abnormal hyperphosphorylation of tau.

Topics: Alzheimer Disease; Animals; Binding Sites; Blotting, Western; Brain; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hippocampus; Immunohistochemistry; L-Lactate Dehydrogenase; Microtubules; Neocortex; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Rats; tau Proteins; Time Factors

The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.

Topics: Actins; Alzheimer Disease; Animals; Biotinylation; Cell Membrane; Cytosol; Immunohistochemistry; Microscopy, Confocal; Microscopy, Electron; Microspheres; Microtubules; Mutagenesis, Site-Directed; Neurons; Okadaic Acid; PC12 Cells; Phosphorylation; Protein Binding; Rats; tau Proteins; Transfection

In Alzheimer's and other neurodegenerative diseases, hyperphosphorylated tau accumulates in affected neuronal and glial cells in the form of paired helical filaments (PHFs). This tau binds antibody AT100, which recognizes the double phosphorylation site (Thr212/Ser214) that is not present in normal biopsy tau. In primary cultures, highly enriched (>98%) in astrocytes of human fetal brain, three polypeptides of 52, 64, and 70 kD showed immunoreactivity with tau antibodies against non-phosphorylated epitopes, accounting for 88, 12, and <1%, respectively, of the total reactivity. All three polypeptides were phosphorylated at the PHF-1 epitope but not at the epitopes Tau-1, 12E8, AT8, and AT100. Treatment of cultures with okadaic acid resulted in apoptosis characterized by the blebbing of the plasma membrane, condensation of nuclear chromatin, and fragmentation of the nucleus. This treatment also resulted in a 3- to 5-fold increase in the content of both tau protein and phosphorylation. The increases were observed in all phosphorylation sites examined, and included the AT100 site. The AT100 site has been proposed to be generated by protein kinase B/Akt and Cdc2. Since okadaic acid can induce an AD-like hyperphosphorylated state of normal tau in primary cultures of human brain cells, a simple cellular model is available permitting study of self-aggregation of tau and phosphorylation events characteristic of neurodegeneration.

Topics: Alternative Splicing; Alzheimer Disease; Antibodies; Apoptosis; Astrocytes; Brain; CDC2 Protein Kinase; Cell Fractionation; Cell Membrane; Cell Nucleus; Cells, Cultured; Chromatin; Cyclic AMP-Dependent Protein Kinases; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Immunoblotting; Immunohistochemistry; Microscopy, Electron; Okadaic Acid; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; tau Proteins; Time Factors; Up-Regulation

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

Although hyperphosphorylated tau is an established feature of Alzheimer's Disease, its role in the disease process is poorly understood, partly because of lack of suitable animal models. We describe the use of living slices of rat hippocampal formation to study tau phosphorylation. Using the AT8 antibody in an ELISA, phosphorylated tau was detected in freshly frozen slices and it increased significantly in slices that were incubated in an electrophysiological recording chamber; the amount detected was greatest when the homogenisation buffer contained phosphatase and kinase inhibitors. The phosphorylated tau content of the slices increased significantly after exposure to the phosphatase 1 and 2A inhibitor okadaic acid (OA) - 1.5 microM. Electrophysiological recordings confirmed that slices were alive and that OA had no acute toxic effect. In control slices phosphorylated tau, detected immunohistochemically, was mainly in the somatodendritic compartment of neurones; in OA treated slices, there was an apparent decrease in somatodendritic AT8 staining and an increase in neuropil staining. Our system enables the induction of hyperphosphorylated tau within living slices, in an experimental environment that can be used to study the biological consequences of such a change, and may therefore help further our understanding of the significance of hyperphosphorylated tau in Alzheimer's Disease.

Topics: Alzheimer Disease; Animals; Bicuculline; Brain Chemistry; Buffers; Electrophysiology; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; GABA Antagonists; Hippocampus; Ionophores; Male; Membrane Potentials; Okadaic Acid; Organ Culture Techniques; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Phosphatase 1; Rats; Rats, Wistar; tau Proteins

Topics: Alzheimer Disease; Animals; Brain Chemistry; Enzyme Inhibitors; Injections, Intraventricular; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Rats; tau Proteins

Topics: Alzheimer Disease; Animals; Brain Damage, Chronic; Enzyme Inhibitors; Injections, Intraventricular; Okadaic Acid; Rats; Rats, Wistar

Intraneuronal aggregation of specific hyperphosphorylated tau isoforms in subsets of neurons may explain many neurodegenerative processes. Only some antibodies including AP422 and AT100 are specific to the abnormal phosphorylation of tau proteins in these processes. AT100-immunoreactivity was never observed in cell models with the exception of Sf9 cells. In the present study, we developed a way to induce AT100-immunoreactivity in different cell types including COS and SY5Y cells after tau cDNA transfection and treatment by okadaic acid. This represents a useful model to study abnormal tau phosphorylation in situ.

Topics: Alzheimer Disease; Animals; Antibodies, Monoclonal; Cell Line, Transformed; COS Cells; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Epitopes; Okadaic Acid; Phosphorylation; tau Proteins; Transfection; Tumor Cells, Cultured

In Alzheimer's disease, tau protein becomes hyperphosporylated, which can contribute to neuronal degeneration. However, the implicated protein kinases are still unknown. Now we report that lithium (an inhibitor of glycogen synthase kinase-3) causes tau dephosphorylation at the sites recognized by antibodies Tau-1 and PHF-1 both in cultured neurons and in vivo in rat brain. This is consistent with a major role for glycogen synthase kinase-3 in modifying proline-directed sites on tau protein within living neurons under physiological conditions. Lithium also blocks the Alzheimer's disease-like proline-directed hyperphosphorylation of tau protein which is observed in neurons treated with a phosphatase inhibitor. These data raise the possibility of using lithium to prevent tau hyperphosphorylation in Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Blotting, Western; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclin-Dependent Kinases; Enzyme Inhibitors; Flavonoids; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Kinetin; Lithium; Neurons; Okadaic Acid; Phosphorylation; Proline; Purines; Rats; tau Proteins

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Bucladesine; Cell-Free System; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cytosol; Enzyme Inhibitors; Ethers, Cyclic; Gene Expression Regulation; Golgi Apparatus; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Okadaic Acid; PC12 Cells; Protein Kinase C; Protein Processing, Post-Translational; Rats

Human tau phosphorylation has been studied in transfected COS-1 cells. Treatment with okadaic acid alters the electrophoretic mobility of human tau protein transiently expressed in transfected cells, due to an increase in the level of phosphorylation. Treatment with okadaic acid also results in an increased phosphorylation of Alzheimer's disease-type phosphoepitopes. Tau phosphorylation within COS-1 cells is partially inhibited by in vivo treatment with DRB, a protein kinase inhibitor. Double treatment of transfected cells with okadaic acid and DRB reveals that phosphorylation of tau protein at the AT8 epitope is achieved by a DRB-resistant protein kinase which is different from that responsible for tau phosphorylation at the SMI-31 epitope, which appears to be sensitive to DRB.

Topics: Alzheimer Disease; Animals; Base Sequence; Cell Line; Chlorocebus aethiops; DNA Primers; Enzyme Inhibitors; Ethers, Cyclic; Humans; Microtubules; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; tau Proteins; Transfection

The microtubule-associated protein tau is more highly phosphorylated at certain residues in developing brain and in Alzheimer's disease paired helical filaments than in adult brain. We examined the regulation of tau phosphorylation at some of these sites in rat brain using the phosphorylation state-dependent anti-tau antibodies AT8, Tau1, and PHF1. The AT8 and PHF1 antibodies bind to phosphorylated tau, while Tau1 binds to unphosphorylated tau. Levels of tau reactive for AT8 were high only during the first postnatal week, with levels in adult declining to approximately 5% of the levels in neonates. In neonatal forebrain slices, tau became rapidly dephosphorylated at the AT8 and Tau1 sites during incubation at 34 degrees C, but was incompletely dephosphorylated at the PHF1 site. Dephosphorylation at AT8 sites, but not at Tau1 or PHF1 sites, was completely inhibited by 1 microM okadaic acid. Hence the regulation of tau phosphorylation by okadaic acid-sensitive phosphatase(s) was site-specific. Addition of 1 microM okadaic acid after dephosphorylation at the AT8 locus yielded a partial recovery of AT8 immunoreactivity, and incubation with basic fibroblast growth factor increased phosphorylation at the AT8 site in a dose-dependent manner. These results indicate that endogenously active and basic fibroblast growth factor stimulated tau kinases directed toward an Alzheimer's disease-related site were present in the slices. In adult brain slices, the small pool of AT8-reactive tau was remarkably insensitive to dephosphorylation during incubation, and okadaic acid treatment induced only small increases in AT8 immunoreactivity. These results suggest that tau phosphorylation in adult brain is less dynamic than in neonatal brain. These findings indicate that neonatal tau is not only phosphorylated more highly than adult tau, but also more dynamically regulated by protein phosphatases and protein kinases than adult tau. The inability of okadaic acid to induce large increases in tau phosphorylation in adult rat brain slices suggests that a loss of protein phosphatase activity alone would not be sufficient to produce the hyperphosphorylation observed in Alzheimer's disease paired helical filaments. Stimulation of kinase activity by basic fibroblast growth factor is likely to modulate tau function during development, and may contribute to the genesis of hyperphosphorylated tau in Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Animals, Newborn; Blotting, Western; Enzyme Inhibitors; Epitopes; Ethers, Cyclic; Female; Fibroblast Growth Factor 2; In Vitro Techniques; Male; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Prosencephalon; Rats; Rats, Sprague-Dawley; tau Proteins

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Topics: Adult; Alzheimer Disease; Base Sequence; Brain; Cell Line; Ethers, Cyclic; Fetal Proteins; Fetus; Humans; Molecular Sequence Data; Okadaic Acid; Phosphorylation; tau Proteins

Aberrant metabolism of the Alzheimer amyloid precursor protein (APP) or its amyloidogenic A beta fragment is thought to be centrally involved in Alzheimer's disease. Nonamyloidogenic processing of APP involves its cleavage within the A beta domain by a protease, termed alpha-secretase, and release of the large extracellular domain, termed APPS. Secretion of APPS can be stimulated by phorbol esters, activators of protein kinase C, with concurrent inhibition of A beta production. While the role of protein kinases of APP metabolism has been investigated, considerably less effort has been devoted to elucidating the role played by protein phosphatases. Okadaic acid, a protein phosphatase inhibitor, has been shown to stimulate secretion of APPS, but the identity of the phosphatase involved has not been investigated.. The secretion of APPS from COS-1 cells was measured in the absence or presence of various doses of serine/threonine-specific phosphatase inhibitors. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase involved in the control of APP secretion.. The availability of protein phosphatase inhibitors with different relative potencies against the different types of serine/threonine-specific protein phosphatase allowed us to examine which of the four known types of protein phosphatase might be involved in the regulation of APP secretion. Both okadaic acid and calyculin A stimulated the secretion of APP from COS-1 cells in a dose-dependent manner. The half-maximal dose for stimulation of APP secretion was approximately 100-fold higher with okadaic acid than with calyculin A.. The nearly 100-fold difference in the observed IC50 values for okadaic acid and calyculin A implicates a type 1 protein phosphatase in the control of APPS production. Protein phosphatase 1 (PP1) is known to be highly expressed in adult mammalian brain, both in neurons and glia. The identification of a specific phosphatase type in the control of APP secretion opens new avenues to the development of rational therapeutic intervention strategies aimed at the prevention and/or treatment of Alzheimer's Disease.

Topics: Aged; Alzheimer Disease; Amyloid; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Cantharidin; Cell Line; Endopeptidases; Enzyme Inhibitors; Ethers, Cyclic; Humans; Immunoblotting; Marine Toxins; Okadaic Acid; Oxazoles; Phorbol 12,13-Dibutyrate; Phosphoprotein Phosphatases; Prion Proteins; Prions; Protein Phosphatase 1; Protein Precursors

In vivo phosphorylation was stimulated in the rat basal nucleus by stereotaxic injection of the phosphatase inhibitor okadaic acid (OA). Hyperphosphorylation of neurofilaments and of the microtubule-associated proteins tau and MAP2 was associated with their redistribution from the axonal compartment into the cell bodies of large projection neurones where they appeared as paired helical filament (PHF)-like immunoreactivity. Astrocytes showed a dramatic increase in APP immunoreactivity and changed their appearance to a stellate shape with long processes. The results demonstrate that abnormal tau phosphorylation and changes in the expression and/or metabolism of APP can be induced in vivo by altering protein phosphorylation. The present experimental paradigm might, therefore, provide a useful tool to model early steps of the pathomechanism of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Astrocytes; Basal Ganglia; Cytoskeleton; Disease Models, Animal; Ethers, Cyclic; Gene Expression Regulation; Male; Microtubule-Associated Proteins; Microtubules; Neurofibrillary Tangles; Okadaic Acid; Phosphorylation; Protein Processing, Post-Translational; Rats; Rats, Wistar; Stereotaxic Techniques; tau Proteins

The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Alzheimer Disease; Amino Acid Sequence; Animals; Brain; Ethers, Cyclic; Hippocampus; Humans; Macaca mulatta; Male; Microtubules; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Rats; Rats, Sprague-Dawley; tau Proteins; Temporal Lobe

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

The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stimulates protein kinase C, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Topics: Adrenal Gland Neoplasms; Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Animals; Cell Line; Ethers, Cyclic; Humans; Kinetics; Methionine; Molecular Weight; Okadaic Acid; Pheochromocytoma; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Biosynthesis; Protein Kinase C; Protein Precursors; Protein Processing, Post-Translational; Tetradecanoylphorbol Acetate; Transcription, Genetic