Okadaic Acid Treatment Research Articles

Exploring Novel GSK-3β Inhibitors for Anti-Neuroinflammatory and Neuroprotective Effects: Synthesis, Crystallography, Computational Analysis, and Biological Evaluation.

In the pathogenesis of Alzheimer's disease, the overexpression of glycogen synthase kinase-3β (GSK-3β) stands out due to its multifaced nature, as it contributes to the promotion of amyloid β and tau protein accumulation, as well as neuroinflammatory processes. Therefore, in the present study, we have designed, synthesized, and evaluated a new series of GSK-3β inhibitors based on the N-(pyridin-2-yl)cyclopropanecarboxamide scaffold. We identified compound 36, demonstrating an IC50 of 70 nM against GSK-3β. Subsequently, through crystallography studies and quantum mechanical analysis, we elucidated its binding mode and identified the structural features crucial for interactions with the active site of GSK-3β, thereby understanding its inhibitory potency. Compound 36 was effective in the cellular model of hyperphosphorylated tau-induced neurodegeneration, where it restored cell viability after okadaic acid treatment and showed anti-inflammatory activity in the LPS model, significantly reducing NO, IL-6, and TNF-α release. In ADME-tox in vitro studies, we confirmed the beneficial profile of 36, including high permeability in PAMPA (Pe equals 9.4) and high metabolic stability in HLMs as well as lack of significant interactions with isoforms of the CYP enzymes and lack of considerable cytotoxicity on selected cell lines (IC50 > 100 μM on HT-22 cells and 89.3 μM on BV-2 cells). Based on promising pharmacological activities and favorable ADME-tox properties, compound 36 may be considered a promising candidate for in vivo research as well as constitute a reliable starting point for further studies.

A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Impaired Dopamine Release and Latent Learning in Alzheimer's Disease Model Zebrafish.

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 (Danio rerio) with okadaic acid (OKA) to model AD and assessed the resulting behavioral and neurochemical changes. We then employed a latent learning paradigm to assess cognitive and motor function followed by neurochemical analysis with fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes to measure the electrically stimulated dopamine release. The behavioral assay showed that OKA treatment caused fish to have lower motivation to reach the goal chamber, resulting in impeded learning and decreased locomotor activity compared to controls. Our voltammetric measurements revealed that the peak dopamine overflow in OKA-treated fish was about one-third of that measured in controls. These findings highlight the profound neurochemical changes that may occur in AD. Furthermore, they demonstrate that applying the latent learning paradigm and FSCV to zebrafish is a promising tool for future neurochemical studies and may be useful for screening drugs for the treatment of AD.

GADD34-mediated dephosphorylation of eIF2\u03b1 facilitates pseudorabies virus replication by maintaining de novo protein synthesis

Viruses have evolved multiple strategies to manipulate their host’s translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

The effects of okadaic acid-treated SH-SY5Y cells on microglia activation and phagocytosis.

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.

Serotonin involvement in okadaic acid-induced diarrhoea in vivo

The consumption of contaminated shellfish with okadaic acid (OA) group of toxins leads to diarrhoeic shellfish poisoning (DSP) characterized by a set of symptoms including nausea, vomiting and diarrhoea. These phycotoxins are Ser/Thr phosphatase inhibitors, which produce hyperphosphorylation in cellular proteins. However, this inhibition does not fully explain the symptomatology reported and other targets could be relevant to the toxicity. Previous studies have indicated a feasible involvement of the nervous system. We performed a set of in vivo approaches to elucidate whether neuropeptide Y (NPY), Peptide YY (PYY) or serotonin (5-HT) was implicated in the early OA-induced diarrhoea. Fasted Swiss female mice were administered NPY, PYY(3–36) or cyproheptadine intraperitoneal prior to oral OA treatment (250 µg/kg). A non-significant delay in diarrhoea onset was observed for NPY (107 µg/kg) and PYY(3–36) (1 mg/kg) pre-treatment. On the contrary, the serotonin antagonist cyproheptadine was able to block (10 mg/kg) or delay (0.1 and 1 mg/kg) diarrhoea onset suggesting a role of 5-HT. This is the first report of the possible involvement of serotonin in OA-induced poisoning.

Neuroprotective Studies of Evodiamine in an Okadaic Acid-Induced Neurotoxicity.

Background: 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 Evodia rutaecarpa, has been reported to ameliorate blood–brain barrier (BBB) permeability and improve cognitive impairment in ischemia and AD mouse models. However, whether evodiamine alleviates tauopathy remains unclear. This study aimed to examine whether evodiamine ameliorates tau phosphorylation and cognitive deficits in AD models. Methods: 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. Results: 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. Conclusions: 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.

CLK inhibition alters tau splicing and has a neuroprotective effect in vitro

AbstractBackgroundMicrotubule‐associated protein tau (MAPT) undergoes alternative splicing at exon 10 which encodes one of its four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau isoforms with all four repeats (4R) whilst exon 10 exclusion leads to isoforms with three repeats (3R). The 1:1 ratio between 3R and 4R isoforms is tightly regulated by splicing machinery and crucial to normal function. Kinases such as CLKs regulate the activity of splicing factors and their interaction with target transcripts, including MAPT. Disruption to this balance leads to the formation of neurofibrillary tangles (NFTs) and neurodegeneration but the mechanism of this is not yet wholly understood.MethodsDifferentiated Neuro2a and SH‐SY5Y cells were used as neuronal models as these cell lines show an imbalance of MAPT isoforms with bias towards 4R and 3R respectively. Splicing factor SRSF1 was transiently knocked down to confirm its role in MAPT splicing. Novel kinase inhibitors were used to reduce the activity of CLKs and determine the effect on MAPT splicing. The expression ratio of 4R and 3R isoforms was measured by RT‐PCR. Okadaic acid (OA) was used to produce a neurotoxic model and investigate the use of kinase inhibitors for neuronal protection. As a functional measure β3‐tubulin fluorescent marker was used to quantify OA dependent reduction in neurite outgrowth.Results4R:3R tau expression significantly decreased by 65% in Neuro2a cells with knockdown of SRSF1 (students t‐test, p<0.001). CLK inhibition increased 4R:3R by 50% in SH‐SY5Y cells compared to control (one‐way ANOVA, p<0.05). SHSY5Y cells neurite outgrowth was significantly reduced by 60% and 85% with 1nM and 5nM OA treatment respectively, demonstrating a concentration dependent effect (one‐way ANOVA, p<0.001). At 1nM OA, neurite outgrowth was rescued by co‐treatment with a novel CLK inhibitor (two‐way ANOVA, p<0.0001).ConclusionsNovel CLK inhibitors can modulate the expression of MAPT splicing isoforms and exert a functional effect on neurite outgrowth. This suggests these compounds could have a therapeutic effect by rectifying the balance of 4R:3R tau expression.

Anti-Inflammatory and Tau Phosphorylation-Inhibitory Effects of Eupatin.

Alzheimer’s disease (AD), which is among the most prevalent neurodegenerative diseases, manifests as increasing memory loss and cognitive decline. Tau phosphorylation and aggregation are strongly linked to neurodegeneration, as well as associated with chronic neuroinflammatory processes. The anti-inflammation effects of natural products have led to wide recognition of their potential for use in treating and preventing AD. This study investigated whether eupatin, a polymethoxyflavonoid found in Artemisia species, has inhibitory effects on neuroinflammation and tau phosphorylation. We treated mouse macrophages and microglia cells with lipopolysaccharides (LPSs) to activate inflammatory signals, and we treated neuronal cells with a protein phosphatase 2A inhibitor, okadaic acid (OA), or transfection with pRK5-EGFP-Tau P301L plasmid to induce tau phosphorylation. The results indicated that eupatin significantly reduced the LPS-induced protein expression and phosphorylation of p65 and inducible nitric oxide synthase as well as downstream products interleukin 6 and nitrite, respectively. Furthermore, eupatin markedly inhibited the expression of phospho-tau in response to OA treatment and plasmid transfection. We discovered that this inhibition was achieved through the inhibition of glycogen synthase kinase 3β (GSK3β), and molecular docking results suggested that eupatin can sufficiently bind to the GSK3β active site. Our results demonstrate that eupatin has neuroprotective effects, making it suitable for AD treatment.

High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation

Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigated whether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitro via activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaic acid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flow cytometry, respectively. Release of HMGB1, TNFα or IL-6 was analyzed by ELISA. Oxidative stress was evaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/ Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac) by Western blot and the association between PP2Ac and α4 by co-immunoprecipitation. Activation of the NF-κB signaling pathway was assessed by detecting Ser32 phosphorylation level of IκBα as well as nuclear entry of p65 protein by Western blot. Activation of the GSK3β/MCL1 signaling pathway was assessed by detecting Ser9 phosphorylation level of GSK3 β and protein level of MCL1. We found Metformin pre-treatment attenuated human and rat cardiomyocytes apoptosis, HMGB1, TNFα and IL-6 release and ROS production that were induced by high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metformin reduced the upregulation of PP2Ac pY307 and the PP2Ac-a4 association, which was not affected by okadaic acid treatment. Metformin pre-treatment reduced NF-κB activation in human and rat cardiomyocytes apoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaic acid treatment. GSK3 β/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition. In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human and rat cardiomyocytes in vitro in a PP2A dependent manner.

Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5′AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.

Okadaic acid promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by inhibiting protein phosphatase 2A.

As a specific inhibitor of serine/threonine protein phosphatases, okadaic acid (OA) has been found to be a tumor promoter. However, whether OA plays a role in metastasis of hepatocellular carcinoma (HCC) has not been well elucidated. In this study, Hep3B and HepG2 cells were treated with different doses of OA and the cell viability was determined by CCK8 test. As a result, Hep3B and HepG2 cells showed no obvious cytotoxicity after OA treatment below 20 or 25 nM for 12 or 24 hours. However, wound healing, invasion, and migration abilities of HCC cells were significantly enhanced in the OA-treated groups than those of the control group (P < .05), measured by cell scratching and BD transwell assays. Moreover, we found that the expression of epithelial-mesenchymal transition (EMT)-related key factors was changed upon OA treatment in a dose-dependent manner. In addition, the activity of protein phosphatase 2A (PP2A) in OA-treated cells was also decreased significantly compared with the control cells (P < .05). Interfering of PP2A subunit A or C caused a similar expression change of EMT-related key factors as the OA treatment in HCC cells. Our results suggest that OA promotes the EMT process of HCC cells by inhibiting the activity of PP2A.

Interaction of the SARS-COV2 Envelope Protein (E) with Lysophosphatidic Acid Receptor 1 (LPAR1) and Additional Human Proteins Involved in Inflammation, Immunity, ADP Ribosylation and Vesicular Transport

A proteomic screen of human proteins interacting with the SARS-COV2 Envelope (E) protein identified LPAR1 as a strong candidate. Physical association of E protein and LPAR1 was confirmed by co-immunoprecipitation and cell surface staining. LPAR1 interaction was confirmed in all eight human cell lines tested. Okadaic acid treatment inhibited binding of the E protein to cell surfaces, suggesting that LPAR1 phosphorylation status may be a factor in the interaction. Many additional proteins, including DMBT1, and proteins involved in endoplasmic reticulum, Golgi, vesicle transport, and ADP ribosylation were also enriched from each of the cell lines, some of which interactions were cell type specific. These findings suggest that LPAR1 is likely a cell surface receptor for the E protein, and pave the way for follow-on studies aimed at understanding the biological significance of the interactions in SARS-COV disease, and elucidating the signaling mechanisms. Funding: VK, ST, and CH were interns at Vistara Bioscience. They were support in part from a Technology Talent Bridge Grant from CTNext. Conflict of Interest: The author declares no conflict of interest.

Subcellular dynamics of variants of SG2NA in NIH3T3 fibroblasts.

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffoldfor multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3β-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3β by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3β caused an initial increase followed by a decrease with a subtle differencein kinetics and intensities. Similar results were also seen upon inhibition of GSK3β by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibitionof extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3β, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus.

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down-regulation of BmPP2A expression. Furthermore, up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down-regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti-BmNPV mechanism, and a possible target for the breeding of silkworm-resistant strains.

Metformin Inhibit Lung Cancer Cell Growth and Invasion in Vitro as Well as Tumor Formation in Vivo Partially by Activating PP2A.

BackgroundThe aim of this study was to investigate whether PP2A activation is involved in the anti-cancer activity of metformin.Material/MethodsA549 and H1651 human lung cancer cells were constructed with stable α4 overexpression (O/E α4) or knockdown of PP2A catalytic subunit A/B(sh-PP2Ac). Influences of okadaic acid (OA) treatment, O/E α4 or sh-PP2Ac on metformin treated cells were investigated by cell viability, proliferation, apoptosis, and Transwell invasion assay in vitro. Protein expression levels of Bax, Bcl-2, Myc, and Akt as well as serine phosphorylation level of Bax, Myc, and Akt were examined by western blot. For in vivo assays, wild type (WT) or modified A549 cells were subcutaneously injected in nude mice, and metformin treatment on these xenografted tumors were assayed by tumor formation assay and western blot detecting cell proliferation marker PCNA (proliferating cell nuclear antigen) as well as protein expression level and serine phosphorylation level of Akt and Myc.ResultsMetformin treatment significantly reduced A549 or H1651 cell growth and invasive capacity in vitro as well as Ser184 phosphorylation of Bax, Ser62 phosphorylation of Myc, and Ser473 phosphorylation of Akt, all of which could be partially attenuated by OA treatment, O/E α4 or sh-PP2Ac. Metformin treatment also significantly reduced tumor formation in vivo as well as protein expression of PCNA, Akt, Myc, and serine phosphorylation of the latter 2, which can be partially blocked by O/E α4 or sh-PP2Ac.ConclusionsMetformin reduced lung cancer cell growth and invasion in vitro as well as tumor formation in vivo partially by activating PP2A.

SUMOylation of periplakin is critical for efficient reorganization of keratin filament network.

The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by small ubiquitin-like modifier 1 (SUMO1). Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper-­SUMOylation of periplakin. Okadaic acid induced hyperphosphorylation-dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous overexpression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs.

Triterpenic Acids from Potentilla parvifolia and Their Protective Effects against Okadaic Acid Induced Neurotoxicity in Differentiated SH-SY5Y Cells.

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.

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.