Okadaic Acid-induced Tau Hyperphosphorylation Research Articles

Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.

Therapeutic Potential of Traditional Oriental Medicines in Targeting Tau Pathology: Insights from Cell-free and Cell-based Screening.

Traditional Oriental Medicines (TOMs) formulated using a variety of medicinal plants have a low risk of side effects. In previous studies, five TOMs, namely Dangguijakyaksan, Hwanglyeonhaedoktang, Ukgansan, Palmijihwanghwan, and Jowiseungchungtang have been commonly used to treat patients with Alzheimer's disease. However, only a few studies have investigated the effects of these five TOMs on tau pathology. This study aimed to examine the effect of five TOMs on various tau pathologies, including post-translational modifications, aggregation and deposition, tau-induced neurotoxicity, and tau-induced neuroinflammation. Immunocytochemistry was used to investigate the hyperphosphorylation of tau induced by okadaic acid. In addition, the thioflavin T assay was used to assess the effects of the TOMs on the inhibition of tau K18 aggregation and the dissociation of tau K18 aggregates. Moreover, a water-soluble tetrazolium-1 assay and a quantitative reverse transcription polymerase chain reaction were used to evaluate the effects of the TOMs on tau-induced neurotoxicity and inflammatory cytokines in HT22 and BV2 cells, respectively. The five TOMs investigated in this study significantly reduced okadaic acid-induced tau hyperphosphorylation. Hwanglyeonhaedoktang inhibited the aggregation of tau and promoted the dissociation of tau aggregates. Dangguijakyaksan and Hwanglyeonhaedoktang attenuated tau-induced neurotoxicity in HT22 cells. In addition, Dangguijakyaksan, Hwanglyeonhaedoktang, Ukgansan, and Palmijihwanghwan reduced tauinduced pro-inflammatory cytokine levels in BV2 cells. Our results suggest that five TOMs are potential therapeutic candidates for tau pathology. In particular, Hwanglyeonhaedoktang showed the greatest efficacy among the five TOMs in cell-free and cell-based screening approaches. These findings suggest that Hwanglyeonhaedoktang is suitable for treating AD patients with tau pathology.

P47phox deficiency improves cognitive impairment and attenuates tau hyperphosphorylation in mouse models of AD

BackgroundAlzheimer’s disease (AD) is characterized by progressive memory loss and cognitive impairment. The aggregation of amyloid β (Aβ) and hyperphosphorylated tau protein are two major pathological features of AD. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) has been indicated in Aβ pathology; however, whether and how it affects tau pathology are not yet clear.MethodsThe role of NOX2 in cognitive function, amyloid plaque formation, and tau hyperphosphorylation were examined in APP/PS1 transgenic mice mated with p47phox-deficient mice (with deletion of the gene of neutrophil cytosolic factor 1, Ncf1) and/or in p47phox-deficient mice receiving intracerebroventricular (ICV) injection of streptozotocin (STZ). The cognitive and non-cognitive functions in these mice were assessed by Morris water maze, Rotarod test, open field, and elevated plus maze. Aβ levels, amyloid plaques, p47phox expression, and astrocyte activation were evaluated using immunofluorescence staining, ELISA, and/or Western blotting. Cultured primary neuronal cells were treated with okadaic acid or conditioned media (CM) from high glucose-stimulated primary astrocytes. The alteration in tau pathology was determined using Western blotting and immunofluorescence staining.ResultsDeletion of the gene coding for p47phox, the organizer subunit of NOX2, significantly attenuated cognitive impairment and tau pathology in these mice. p47phox deficiency decreased the activation of astrocytes but had no effect on Aβ levels and amyloid plaque formation in the brains of aged APP/PS1 mice, which displayed markedly increased expression of p47phox in neurons and astrocytes. Cell culture studies found that neuronal p47phox deletion attenuated okadaic acid-induced tau hyperphosphorylation at specific sites in primary cultures of neurons. CM from high glucose-treated WT astrocytes increased tau hyperphosphorylation in primary neurons, whereas this effect was absent from p47phox-deficient astrocytes.ConclusionsThese results suggest that p47phox is associated with cognitive function and tau pathology in AD. p47phox expressed in neurons contributes to tau hyperphosphorylation directly, while p47phox in astrocytes affect tau hyperphosphorylation by activating astrocytes indirectly. Our results provide new insights into the role of NOX2 in AD and indicate that targeted inhibition of p47phox may be a new strategy for the treatment of AD.

Okadaic acid–induced tau hyperphosphorylation and the downregulation of Pin1 expression in primary cortical neurons

Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.

Neuroprotective effect of paeoniflorin on okadaic acid-induced tau hyperphosphorylation via calpain/Akt/GSK-3β pathway in SH-SY5Y cells

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.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+ concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca2+/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Adenosine A2A receptor inactivation alleviates early-onset cognitive dysfunction after traumatic brain injury involving an inhibition of tau hyperphosphorylation

Tau is a microtubule-associated protein, and the oligomeric and hyperphosphorylated forms of tau are increased significantly after neurotrauma and considered important factors in mediating cognitive dysfunction. Blockade of adenosine A2A receptors, either by caffeine or gene knockout (KO), alleviates cognitive dysfunction after traumatic brain injury (TBI). We postulated that A2AR activation exacerbates cognitive impairment via promoting tau hyperphosphorylation. Using a mouse model of moderate controlled cortical impact, we showed that TBI induced hyperphosphorylated tau (p-tau) in the hippocampal dentate gyrus and spatial memory deficiency in the Morris water maze test at 7 days and 4 weeks after TBI. Importantly, pharmacological blockade (A2AR antagonist ZM241385 or non-selective adenosine receptor antagonist caffeine) or genetic inactivation of A2ARs reduced the level of tau phosphorylation at Ser404 and alleviated spatial memory dysfunction. The A2AR control of p-tau is further supported by the observations that a KO of A2AR decreased the activity of the tau phosphorylation kinases, glycogen synthase kinase-3β (GSK-3β) and protein kinase A (PKA) after TBI, and by that CGS21680 (A2AR agonist) exacerbated okadaic acid-induced tau hyperphosphorylation in cultured primary hippocampal neurons. Lastly, CGS21680-induced neuronal tau hyperphosphorylation and axonal injury were effectively alleviated by individual treatments with ZM241385 (A2AR antagonist), H89 (PKA antagonist) and SB216763 (GSK-3β antagonist), or by the combined treatment with H89 and SB216763. Our findings suggest a novel mechanism whereby A2AR activation triggers cognitive dysfunction by increasing the phosphorylation level of tau protein after TBI and suggest a promising therapeutic and prophylactic strategy by targeting aberrant A2AR signaling via tau phosphorylation.

SLM, a novel carbazole-based fluorophore attenuates okadaic acid-induced tau hyperphosphorylation via down-regulating GSK-3β activity in SH-SY5Y cells

Phosphorylated tau dissociates from microtubules and aggregates to form neurofibrillary tangles resulting in neuronal toxicity and cognitive deficits. Attenuating tau hyperphosphorylation is considered as an effective therapeutic approach for Alzheimer's disease (AD). From our previous study, SLM, a carbazole-based fluorophore prevents Aβ aggregation, reduced glycogen synthase kinase-3β (GSK-3β) activity and tau hyperphosphorylation in triple transgenic mouse model of AD. However, the mechanism by which SLM attenuates tau hyperphosphorylation warrants further investigation. In the current study, we intend to evaluate the effects of SLM against okadaic acid (OA)-induced tau hyperphosphorylation and microtubules instability in human neuroblastoma (SH-SY5Y) cells. The results showed that, SLM reduced the OA-induced cell neurotoxicity and tau hyperphosphorylation in SH-SY5Y cells. SLM treatment down-regulated GSK-3β activity. However, in the presence of GSK-3β inhibitor (SB216763, 10μM), SLM treatment could not reduce GSK-3β activity and tau hyperphosphorylation as compared with SB216763 treatment alone. Furthermore, SLM treatment also ameliorated OA-induced microtubules instability and cytoskeleton damage. Collectively, SLM attenuated OA-induced tau hyperphosphorylation via down-regulating GSK-3β activity in SH-SY5Y cells. Therefore, this study supports SLM as a potential compound for AD and other tau pathology-related neurodegenerative disorders.

The effect of okadaic acid–induced tau hyperphosphorylation on Pin1 expression in primary cortical neurons

The effect of okadaic acid–induced tau hyperphosphorylation on Pin1 expression in primary cortical neurons

Effects of Ginkgolide A on Okadaic Acid-induced Tau Hyperphosphorylation and the PI3K-Akt Signaling Pathway in N2a Cells

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.

Valproate reduces tau phosphorylation via cyclin-dependent kinase 5 and glycogen synthase kinase 3 signaling pathways

Valproate (VPA) is a widely used anticonvulsant and mood-stabilizing drug. Recent studies have shown that VPA could reduce amyloid-β generation, and improve memory deficits in transgenic mouse models of Alzheimer's disease (AD). However, whether VPA affects tau phosphorylation and the underlying mechanism has not been established. Here, we showed that systemic treatment of APP and presenilin 1 double transgenic mice with VPA (50 mg/kg, once a day for 12 weeks), significantly reduced the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. Meanwhile, VPA treatment markedly reduced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), two protein kinases involved in abnormal hyperphosphorylation of tau. In an okadaic acid-induced tau hyperphosphorylation SH-SY5Y cell model, the anti-tau-phosphorylation effect of VPA was further confirmed, accompanied by a marked decrease in the activities of CDK5 and GSK3β. Our present data suggest that the inhibitory effects of VPA on tau hyperphosphorylation might be mediated through both CDK5 and GSK3β signaling pathways.

Acetyl-L-Carnitine Attenuates Okadaic Acid Induced Tau Hyperphosphorylation and Spatial Memory Impairment in Rats

Tau hyperphosphorylation and memory deficit are characteristic alterations of Alzheimer's disease (AD). Protein phosphatases (PP) 2A plays a crucial role in AD-like lesions. Inhibition of PP2A through hippocampal injection of okadaic acid (OA) induces tau hyperphosphorylation and memory impairment of rats. By using this model, we explored in the present study the effects of acetyl-L-carnitine (ALCAR), a constituent of the inner mitochondrial membrane, on the memory retention, tau phosphorylation, and oxidative stress in rats. We found that pre-treatment of ALCAR (50 mg/d . rat, per os) for two weeks efficiently improved the OA-induced spatial memory retention impairment of the rats. ALCAR antagonized tau hyperphosphorylation at multiple AD sites and it abated the OA-induced PP2A inhibition and oxidative stress. Our study provides the first in vivo evidence that ALCAR can attenuate AD-like PP2A inhibition, tau hyperphosphorylation, and spatial memory deficit of the rats. It suggests that ALCAR may hold potential in AD treatment.

Inhibition of PHF-like Tau Hyperphosphorylation in SH-SY5Y Cells and Rat Brain Slices by K252a

Abnormal hyperphosphorylation of tau is believed to constitute a critical biochemical event in the process of neurofibrillary degeneration of Alzheimer's disease. We have developed a cellular model where apparently authentic PHF-like tau hyperphosphorylation is induced by okadaic acid. To gain deeper insight into the complex mechanisms of this pathological process we tested a variety of kinase inhibitors in this model. We found that K252a is differentiated from staurosporine by its inhibition of ERK2: both compounds are structurally related microbial metabolites generally believed to have only moderate kinase selectivity. However, since ERK2 inhibitors are exceedingly rare, we used this differential inhibitory property of K252a to demonstrate the involvement of ERK2 in PHF-type tau hyperphosphorylation. K252a was uniquely able to completely suppress the okadaic acid-induced tau hyperphosphorylation in SH-SY5Y cells and rat brain slices by way of including ERK2 in its inhibitory spectrum, and to conserve the normal binding of tau to tubulin. GSK3 inhibitors partially affected the normal state of tau phosphorylation in SH-SY5Y cells, but had no impact on okadaic acid-induced tau hyperhosphorylation. As K252a is the first molecule identified capable of preventing the spectrum of PHF-like tau hyperphosphorylation markers, it may represent a conceptual starting point for therapeutic development of suitable spectrum kinase inhibitors.

Okadaic acid induces hyperphosphorylation of tau independently of mitogen-activated protein kinase activation.

Hyperphosphorylation of the microtubule-associated protein tau is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on tau common to Alzheimer's disease. Using an okadaic acid-induced tau hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor ¿PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]¿ of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust tau hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in tau phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce tau hyperphosphorylation.