Glycogen Synthase Kinase-3 Levels Research Articles

Cloperastine Reduces IL-6 Expression via Akt/GSK3/Nrf2 Signaling in Monocytes/Macrophages and Ameliorates Symptoms in a Mouse Sepsis Model Induced by Lipopolysaccharide.

Cloperastine (CLP) is a drug with a central antitussive effect that is used to treat bronchitis. Therefore, we have attempted to examine the anti-inflammatory effects of CLP. CLP reduced the secretion of interleukin (IL)-6, a pro-inflammatory cytokine, from RAW264.7 monocyte/macrophage-linage cells treated with lipopolysaccharide (LPS). IL-6 is a biomarker of sepsis and has been suggested to exacerbate its symptoms. We found that the intraperitoneal administration of CLP reduced IL-6 levels in the lungs and also improved hypothermia in mice with LPS-induced sepsis. CLP ameliorated kidney pathologies such as congestion and increased the survival rate of mice administered with a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. CLP induced the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3) and also increased the amount of nuclear factor erythroid-2-related factor 2 (Nrf2) in RAW264.7 cells with/without LPS. Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), reduced the upregulated phosphorylation levels of Akt and GSK3 and the increased amount of Nrf2. It also halted the reduction of IL-6 secretion caused by CLP. These results suggest that CLP has an anti-inflammatory function via Akt/GSK3/Nrf2 signaling and could be a candidate drug for the treatment of inflammatory diseases, including sepsis.

Differential diagnosis of unipolar versus bipolar depression by GSK3 levels in peripheral blood: a pilot experimental study

BackgroundThe differential diagnosis of patients presenting for the first time with a depressive episode into unipolar disorder versus bipolar disorder is crucial to establish the correct pharmacological therapy (antidepressants vs mood stabilizers), but no biological markers are currently available. Several lines of evidence indicate an involvement of Glycogen Synthase Kinase-3 (GSK3) in the pathophysiology of depression. However, previous reports about GSK3 in peripheral blood were incomplete or inconsistent, so a specific marker is not yet available. The aim was to search for consistent differences in GSK3α and GSK3β or of their phosphorylated forms in samples of peripheral blood from patients with unipolar and bipolar depression.MethodsMononucleate peripheral blood cells (PBMCs) of samples from patients presenting with a depressive episode were analyzed with the western blot technique.ResultsThe total amount of GSK3β in PBMCs was significantly lower in patients with bipolar disorder than in patients with unipolar depression. The sensitivity based on GSK3β was 85%. GSK3α was not significantly different but allowed a correct detection of 57% of BD patients. The combination in series of GSK3β and GSK3α yields a sensitivity of about 100%, but with 26.7% false negatives.ConclusionsOur results suggest that PBMC GSK3β could be a candidate biomarker for the differential diagnosis of bipolar disorder versus unipolar depression. This finding may help in implementing the still limited panel of peripheral biomarkers for differential diagnosis between unipolar and bipolar disorder in patients presenting with a depressive episode.

Clinical significance of glycogen synthase kinase 3 (GSK-3) expression and tumor budding grade in colorectal cancer: Implications for targeted therapy

IntroductionGlycogen synthase kinase 3 (GSK-3) has been proposed as a novel cancer target due to its regulating role in both tumor and immune cells. However, the connection between GSK-3 and immunoevasive contexture, including tumor budding (TB) has not been previously examined. Methods: we investigated the expression levels of total GSK-3 as well as its isoforms (GSK-3β and GSK-3α) and examined their potential correlation with TB grade and the programmed cell death-ligand 1 (PD-L1) in colorectal cancer (CRC) tumor samples. Additionally, we compared the efficacy of GSK-3-inhibition with PD-1/PD-L1 blockade in humanized patient-derived (PDXs) xenografts models of high-grade TB CRC. Resultswe show that high-grade (BD3) TB CRC is associated with elevated expression levels of total GSK-3, specifically the GSK-3β isoform, along with increased expression of PD-L1 in tumor cells. Moreover, we define an improved risk stratification of CRC patients based on the presence of GSK-3+/PD-L1+/BD3 tumors, which are associated with a worse prognosis. Significantly, in contrast to the PD-L1/PD-1 blockade approach, the inhibition GSK-3 demonstrated a remarkable enhancement in the antitumor response. This was achieved through the reduction of tumor buds via necrosis and apoptosis pathways, along with a notable increase of activated tumor-infiltrating CD8+ T cells, NK cells, and CD4- CD8- T cells. Conclusionsour study provides compelling evidence for the clinical significance of GSK-3 expression and TB grade in risk stratification of CRC patients. Moreover, our findings strongly support GSK-3 inhibition as an effective therapy specifically targeting high-grade TB in CRC.

Mesenchymal Stem Cells and Begacestat Mitigate Amyloid-β 25-35-Induced Cognitive Decline in Rat Dams and Hippocampal Deteriorations in Offspring.

Alzheimer's disease (AD) is the most common cause of age-related neurodegeneration and cognitive decline. AD more commonly occurs in females than in males, so it is necessary to consider new treatments specifically targeting this population. The present study investigated the protective effects of Begacestat (γ-secretase inhibitor-953, GSI-953) and bone marrow-derived mesenchymal stem cells (BM-MSCs) during pregnancy on cognitive impairment in rat dams and neurodegeneration in offspring caused by the intracerebroventricular injection of Aβ 25-35 before pregnancy. The performances of dams injected with amyloid-β 25-35 (Aβ 25-35) during behavioral tests were significantly impaired. The offspring of Aβ 25-35-injected dams treated with BM-MSCs or GSI-953 showed a dramatically reduced number and size of activated microglial cells, enhancement in the processes length, and a decrease in the proinflammatory cytokine levels. Additionally, BM-MSC or GSI-953 therapy reduced Aβ 25-35-induced increases in tau phosphorylation and amyloid precursor protein levels in the neonates' hippocampus and elevated the lower levels of glycogen synthase kinase-3 and brain-derived neurotrophic factor; moreover, reversed Aβ 25-35-induced alterations in gene expression in the neonatal hippocampus. Finally, the treatments with BM-MSC or GSI-953 are globally beneficial against Aβ 25-35-induced brain alterations, particularly by suppressing neural inflammation, inhibiting microglial cell activation, restoring developmental plasticity, and increasing neurotrophic signaling.

Role of Glycogen Synthase Kinase 3 in Molecular Pathology of Alzheimer’s Disease

Background: Glycogen synthase kinase 3 (GSK-3) is the evolutionary well-preserved multifunctional ubiquitously expressed kinase. In brain, GSK-3 mediates its effects via cascade of intra-cellular signalling pathways that regulate several functions including memory, behaviour, synapse plasticity, bioenergetics, and neuronal fate determination. Several evidences on transgenic mice models and reports from the post-mortem of AD brains posit that altered levels of GSK-3 are closely linked with several pathological features including impaired splitting of amyloid precursor protein, hyperphosphorylation of Tau, mitochondrial dysfunctions, impaired energetics, maladaptive plasticity of neuronal circuitries in dementia, culminating into pathology of Alzheimer’s disease along with other neurodegenerative diseases. Aim & Objective: Present paper has an aim to analyse the role of GSK3b in molecular pathology of Alzheimer’s disease. The involvement of dysregulated GSK3b in the pathophysiology of Alzheimer’s disease is discussed in the critical review paper covering several factors that either contribute to GSK3b dysregulation or interact with dysregulated GSK3b in the pathogenesis of AD. Research Methodology: Critical analytic, qualitative cum retrospective research study design is adopted utilizing secondary data from books, monographs, journals, conference proceedings for the critical evaluation leading to conclusions beneficial either in future research study or in understanding intricate molecular events for pharmaceutical intervention either to ameliorate the clinical manifestations of Alzheimer’s disease or to delays the progression of disease for the benefit of patients with AD. Findings/Result: Several stressors induce overexpression/aberrant activity of GSK3b leading to increased Amyloid beta formation, tau phosphorylation, mitochondrial dysfunction, impaired synaptic activity, release of pro-inflammatory cytokines and other manifestations implicated in the molecular pathology of Alzheimer’s disease. Originality of Paper: Comprehensive approach was adopted to include papers related to the topic within frame-work of inclusion and exclusion criteria to deduce conclusion. Paper Type: Critical analytic review paper.

Neuroprotective Properties of Minocycline Against Methylphenidate-Induced Neurodegeneration: Possible Role of CREB/BDNF and Akt/GSK3 Signaling Pathways in Rat Hippocampus

Neurodegeneration is a side effect of methylphenidate (MPH), and minocycline possesses neuroprotective properties. This study aimed to investigate the neuroprotective effects of minocycline against methylphenidate-induced neurodegeneration mediated by signaling pathways of CREB/BDNF and Akt/GSK3. Seven groups of seventy male rats were randomly distributed in seven groups (n = 10). Group 1 received 0.7ml/rat of normal saline (i.p.), and group 2 was treated with MPH (10mg/kg, i.p.). Groups 3, 4, 5, and 6 were simultaneously administered MPH (10mg/kg) and minocycline (10, 20, 30, and 40mg/kg, i.p.) for 21days. Minocycline alone (40mg/kg, i.p.) was administrated to group 7. Open field test (OFT) (on day 22), forced swim test (FST) (on day 24), and elevated plus maze (on day 26) were conducted to analyze the mood-related behaviors; hippocampal oxidative stress, inflammatory, and apoptotic parameters, as well as the levels of protein kinase B (Akt-1), glycogen synthase kinase 3 (GSK3), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF), were also assessed. Furthermore, localization of total CREB, Akt, and GSK3 in the DG and CA1 areas of the hippocampus were measured using immunohistochemistry (IHC). Histological changes in the mentioned areas were also evaluated. Minocycline treatment inhibited MPH-induced mood disorders and decreased lipid peroxidation, oxidized form of glutathione (GSSG), interleukin 1 beta (IL-1β), alpha tumor necrosis factor (TNF-α), Bax, and GSK3 levels. In the contrary, it increased the levels of reduced form of glutathione (GSH), Bcl-2, CREB, BDNF, and Akt-1 and superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in the experimental animals' hippocampus. IHC data showed that minocycline also improved the localization and expression of CREB and Akt positive cells and decreased the GSK3 positive cells in the DG and CA1 regions of the hippocampus of MPH-treated rats. Minocycline also inhibited MPH-induced changes of hippocampal cells' density and shape in both DG and CA1 areas of the hippocampus. According to obtained data, it can be concluded that minocycline probably via activation of the P-CREB/BDNF or Akt/GSK3 signaling pathway can confer its neuroprotective effects against MPH-induced neurodegeneration.

Mood Regulatory Actions of Active and Sham Nucleus Accumbens Deep Brain Stimulation in Antidepressant Resistant Rats.

The antidepressant actions of deep brain stimulation (DBS) are associated with progressive neuroadaptations within the mood network, modulated in part, by neurotrophic mechanisms. We investigated the antidepressant-like effects of chronic nucleus accumbens (NAc) DBS and its association with change in glycogen synthase kinase 3 (GSK3) and mammalian target of rapamycin (mTOR) expression in the infralimbic cortex (IL), and the dorsal (dHIP) and ventral (vHIP) subregions of the hippocampus of antidepressant resistant rats. Antidepressant resistance was induced via daily injection of adrenocorticotropic hormone (ACTH; 100 μg/day; 15 days) and confirmed by non-response to tricyclic antidepressant treatment (imipramine, 10 mg/kg). Portable microdevices provided continuous bilateral NAc DBS (130 Hz, 200 μA, 90 μs) for 7 days. A control sham electrode group was included, together with ACTH- and saline-treated control groups. Home cage monitoring, open field, sucrose preference, and, forced swim behavioral tests were performed. Post-mortem levels of GSK3 and mTOR, total and phosphorylated, were determined with Western blot. As previously reported, ACTH treatment blocked the immobility-reducing effects of imipramine in the forced swim test. In contrast, treatment with either active DBS or sham electrode placement in the NAc significantly reduced forced swim immobility time in ACTH-treated animals. This was associated with increased homecage activity in the DBS and sham groups relative to ACTH and saline groups, however, no differences in locomotor activity were observed in the open field test, nor were any group differences seen for sucrose consumption across groups. The antidepressant-like actions of NAc DBS and sham electrode placements were associated with an increase in levels of IL and vHIP phospho-GSK3β and phospho-mTOR, however, no differences in these protein levels were observed in the dHIP region. These data suggest that early response to electrode placement in the NAc, irrespective of whether active DBS or sham, has antidepressant-like effects in the ACTH-model of antidepressant resistance associated with distal upregulation of phospho-GSK3β and phospho-mTOR in the IL and vHIP regions of the mood network.

Platelet-Rich Plasma Suppresses Inflammation Reaction of Rat Articular Chondrocytes via Wingless-Related Integration Site/β-Catenin Axis

Our study aims to elucidate the role of platelet-rich plasma (PRP) in rats chondrocytes inflammation and mechanism. PRP was obtained from 8 weeks old rats. Then, the knee joint of bilateral hind limbs was dissected and articular chondrocytes were obtained in super-clean table after dislocation and identified at the second generation during culture and passage. Chondrocytes were divided into control group 1 (addition of saline), control group 2 (IWP-2, Wnt/β-catenin axis inhibitor) and experimental group (PRP) followed by analysis of mRNA levels of glycogen synthase kinase-3 (GSK-3β), low-density lipoprotein receptor-associated protein 5 (LRP5), Wnt1 and β-catenin by RT-PCR, IL-1 and TNF-α after 1 week by ELISA. The second generation articular chondrocytes presented polygonal or triangular cell morphology, positive for collagen II and toluidine blue staining. PRP addition significantly reduced GSK-3β and LRP5 mRNA level, and increased β-catenin and Wnt1 mRNA levels in chondrocytes. Meanwhile, it suppressed IL-1 and TNF-α secretion and Wnt protein production inhibitor 2. PRP might suppresses inflammatory factors production of rat articular chondrocytes through inhibiting Wnt/β-catenin axis.

The role of GSK-3 in mood disorders: Preliminary data from an experimental study

IntroductionThe identification of potential biomarkers is crucial to improve the management and treatment of mood disorders. Glycogen synthase kinase-3 (GSK-3) is a multifunctional enzyme with an important role in the etiology of mood disorders. Recent findings suggested GSK-3 as a putative biomarker in mood disorders.ObjectivesThe aims of the study are: - to evaluate GSK3 as potential biomarker for differential diagnosis (MDD and BD); - to analyze the regulation of GSK3 by psychopharmacological treatments.MethodsPatients included fulfill the following criteria: (a) principal diagnosis of MDD or BD (DSM-5); (b) age ≥ 18 years; (c) drug-free for at least 4 weeks before the inclusion. For each patient included a healthy control is enrolled, matched by gender and age. All included subjects at the study entry point (t0) are assessed through: - semistructured clinical interview and clinical rating scales (Hamilton Depression Rating Scale, Hamilton Anxiety Rating Scale; Young Mania Rating Scale, Clinical Global Impression) - blood draw, to measure GSK-3 levels Patients with MDD or BD are assessed again after 1 week (T1) and after 2 month (T2) of specific pharmacological treatment.ResultsSo far, we enrolled 16 patients and 16 healthy controls. The enrollment is still ongoing.ConclusionsWe expect to find GSK-3 levels differently expressed between healthy controls, patients with DDM and patients with BD. This finding would be crucial as it could contribute to the improvement of differential diagnosis. Moreover, we expect to observe a change in GSK-3 levels after psychopharmacological treatments.

Insulin receptors regulate the fecundity of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Abstract Two InR (insulin receptor) genes have been identified in the Nilaparvata lugens. In this study, we used RNA interference (RNAi) to investigate the role of InR genes in the fecundity of N. lugens. The expression of NLInR1 and NLInR2 genes was simultaneously silenced with mixture of dsInR1 and dsInR2 (dsInRs) injection. Our results showed that larvae RNAi against both NLInR1 and NLInR2 reduced the number of eggs laid by N. lugens and some eggs as well as ovaries were abnormal. In addition, the relative expression of Vg (vitellogenin) and VgR (vitellogenin receptor) was significantly reduced on the 4th and 6th days after insects treated with larvae RNAi reached the adult stage. We also determined the relative expression levels of insulin/insulin-like signaling (IIS) related genes in RNAi-treated larvae and found that the expression levels of Chico (homologous receptor substrate), Akt (protein kinase B), PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog) genes decreased whereas FOXO (forkhead box O) and GSK3 (glycogen synthase kinase-3) levels increased on the 4th and 6th days after insects reached the adult stage. These results indicate that silencing of NLInR1 and NLInR2 reduces the fecundity of N. lugens through the IIS pathway.

The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis.

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/β were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/β after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/β. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

Drospirenone-containing contraceptive exerts positive effects on cardiac uric acid and PAI-1 but not GSK-3: Improved safety profiles in contraception?

The use of combined oral contraceptives (COC) have been associated with increased risk of adverse cardiovascular events and elevated cardiac and circulating plasminogen activator inhibitor-1 (PAI-1) and glycogen synthase kinase-3 (GSK-3) have been implicated in these events. Contraceptives containing drospirenone, a progestin with anti-androgenic actions may have a positive or neutral effect on cardiac PAI-1 and GSK-3 levels. Studies on the favorable effects of oral contraceptives containing drospirenone when compared with other androgenic contraceptives have not been fully elucidated. We therefore sought to compare the effect of a contraceptive containing ethinyl estradiol and drospirenone (DSP) with a contraceptive containing ethinyl estradiol and levonorgestrel (LVG) on cardiac uric acid (UA), PAI-1, GSK-3 and some hematological parameters. Ten weeks old female Wistar rats were divided into three groups; control (CON), LVG or DSP treated rats. The treatment lasted for 8 weeks. Results showed that LVG and not DSP treatment led to increase in plasma and cardiac tissue UA, plasma and cardiac PAI-1 as well as granulocyte-lymphocyte ratio (GLR) and platelet-lymphocyte ratio (PLR). However, the DSP treatment affected the circulating GSK-3. Taken together, the findings showed that LVG and not DSP affected cardiac UA and PAI-1. These results suggest that COC containing drospirenone appears to have positive effects on cardiac UA and PAI-1 levels but do not affect GSK-3, hence, COC containing drospirenone may be a better and safer means of contraception compared to androgenic contraceptives.

GSK-3\u03b2 inhibits autophagy and enhances radiosensitivity in non-small cell lung cancer

BackgroundRadiotherapy is one of the most common and effective treatment methods for cancer, and improving the radiosensitivity of tumor tissues during the treatment process is vital. We report the mechanisms of glycogen synthase kinase 3 (GSK-3) β-regulated autophagy and the effects of autophagy on radiosensitivity in non-small cell lung cancer (NSCLC).MethodImmunohistochemical staining was performed to determine GSK-3β tissue expression in 89 NSCLC patients with follow-up data and the expression status of GSK-3β and autophagy in NSCLC tissues after X-ray radiotherapy. Western blots were used to quantitate changes in autophagy-related protein expression after A549 cells were treated with GSK-3β inhibitors and after H460 cells were transfected with GSK-3β mutants with different activities and X-ray irradiated. Clonogenic assays were used to measure the effect of autophagy on cellular proliferation.ResultsGSK-3β expression positively correlated with NSCLC differentiation (P < 0.05), and GSK-3β negativity was associated with a better prognosis in 89 NSCLC patients. After X-ray irradiation, the expression levels of GSK-3β and p62 were decreased in NSCLC tissues, and the expression levels of the autophagy-related protein LC3 were increased. A549 and H460 cells were selected as representative GSK-3β-high and GSK-3β-low expression cell lines. After transfecting H460 cells with different GSK-3β mutants [wild type GSK-3β (GSK-3β-WT), constitutively active GSK-3β (GSK-3β-S9A), and catalytically inactive GSK-3β (GSK-3β-K85R)] and subjecting these cells to X-ray irradiation, AMPK and LC3 expression levels decreased, and p62 expression levels increased. These effects were particularly significant for the GSK-3β-S9A mutant. In A549 cells, after GSK-3β inhibition and X-ray irradiation, AMPK and LC3 protein expression levels increased. Moreover, when autophagy was inhibited, cell proliferation decreased.ConclusionOur studies revealed that GSK-3β expression is associated with NSCLC differentiation, and patients with GSK-3β-negative tumors had a better prognosis. X-ray irradiation inhibited GSK-3β expression and promoted autophagy. Therefore, GSK-3β inhibits autophagy and enhances the radiosensitivity of NSCLC cells.

Gestational glucocorticoid exposure disrupts glucose homeostasis that is accompanied by increased endoglin and DPP-4 activity instead of GSK-3 in rats

Gestational glucocorticoid (GC) treatment has been associated with cardiometabolic disorder (CMD) in offspring’s in later life. Elevated dipeptidyl peptidase-4 (DPP-4) activity, endoglin and glycogen synthase kinase-3 (GSK-3) has also been implicated in the development of insulin resistance (IR) and/or vascular inflammation. We aimed to investigate the impact of GC exposure on glucose metabolism and the circulating levels of inflammatory biomarkers, DPP-4 activity and GSK-3 in pregnant rats. Pregnant Wistar rats received either vehicle or dexamethasone (DEX; 0.2 mg/kg; po) between gestational days 14 and 19. Gestational GC exposure resulted in impaired glucose homeostasis that is accompanied with elevated circulating levels of inflammatory biomarkers (endoglin, uric acid, and platelet/lymphocyte ratio), oxidative stress (malondialdehyde), blood viscosity, reduced NO level and increased DPP-4 activity. However, these effects were associated with atherogenic dyslipidemia and reduced GSK-3.We conclude that plasma endoglin, a marker of vascular inflammation, and plasma DPP-4 activity are increased in pregnant rats treated with GC during late gestation. Therefore, glucose deregulation associated with gestational GC exposure is through endoglin-/DPP-4-dependent but GSK-3-independent pathway.

Mapping Protein:Protein Interaction of the FGF14:GSK3β Complex

Glycogen synthase kinase 3 (GSK3) is a multifaceted enzyme with ubiquitous expression in the central nervous system (CNS). Increased levels of GSK3 trigger a cascade of serine/threonine (S/T) phosphorylation events that correlates with maladaptive plasticity of neuronal circuitries in neuropsychiatric disorders spurring an interest in identifying new molecular targets within this pathway for drug discovery purposes. In previous studies we have shown that changes in GSK3 levels modify neuronal excitability by regulating protein:protein interactions (PPI) between fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ channel. Suppression of GSK3 leads to loss in neuronal excitability in hippocampal neurons with dispersion of the FGF14:Nav channel complex from the axonal initial segment. However, whether GSK3 phosphorylates FGF14 or Nav channels or forms a stable complex with any of these two proteins remains to be determined. Building on our in vitro phosphorylation and mass spectrometry studies demonstrating that GSK3β phosphorylates FGF14 at S226, we posited that GSK3β could form a PPI complex with FGF14. To test this hypothesis, we apply Surface Plasmon Resonance (SPR) on E.coli purified proteins to assess putative binding affinity of FGF14 to GSK3β and map interaction sites. SPR studies revealed strong binding affinity (kD=0.159 µM) between FGF14 and GSK3β pointing for the C-tail of FGF14 as a necessary structural element mediating PPI. Overall, these studies identify new critical structural determinants of the FGF14:GSK3 complex that might serve as a base for therapeutic development against GSK3-related brain disorders.

Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

Glycogen Synthase Kinase-3 Modulates Hyperosmotic-Induced Urea Transporter A1 Relocation in the Inner Medullary Collecting Duct Cells

Aim: Glycogen synthase kinase 3 (GSK3) regulates urine concentration by mediating the vasopressin-induced aquaporin 2 expression and water permeability, although it is unknown whether GSK3 also mediates the accumulation of the urea transporter A1 (UT-A1). The aim of this study is to investigate the effect of GSK3 on UT-A1 distribution. Methods: Mouse inner medullary collecting duct 3 cells were transfected with UT-A1-GFP construct. The stable transfected cells were cultured under hypertonic conditions, treated with GSK3 inhibitor lithium chloride, GSK3 activator, lysosome or proteasome inhibitor. The expression levels of UT-A1, GSK3, and phospho-GSK3 were analyzed using western blot. The interaction between UT-A1 and the Golgi apparatus was examined using confocal immunofluorescence microscope. The UT-A1 trafficking was examined using the biotinylation of surface membranes. Results: UT-A1 dissociated away from the Golgi apparatus and translocated to the plasma membrane under hypertonic-NaCl and NaCl plus urea stimulation. This movement was accompanied by the increased phosphorylation of GSK3 and its localization on the cellular membrane. Moreover, these results were duplicated by treating the cells with the GSK3 inhibitor, and by contrast, were partially reversed by the GSK3 activator. Treating cells with a lysosome or proteasome inhibitor failed to attenuate the effects of hypertonic stimulus, indicating that the loss of UT-A1 from the Golgi was not due to degradation. Conclusion: Our results suggest that GSK3 may in part modulate the hypertonic-induced intracellular UT-A1 redistribution and its accumulation on the plasma membrane, which may constitute another mechanism by which GSK3 modulates urine concentration.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

Natural Functions of PLIN2 Mediating Wnt/LiCl Signaling and Glycogen Synthase Kinase 3 (GSK3)/GSK3 Substrate-Related Effects Are Modulated by Lipid.

Belonging to the PLIN family, PLIN2 associates with lipid storage droplets (LSDs), but other functions of PLIN2 remain unclear. Here, we suggest that PLIN2 mediates Wnt signaling because PLIN2 small interfering RNA (siRNA) suppresses activation of Wnt/coreceptor pathways. The mediation in the Wnt/Frizzled pathway seems to occur from Dishevelleds to axin/glycogen synthase kinase 3(GSK3)/β-catenin complexes (AGβC) as Wnt decreases Dishevelled/PLIN2 but increases AGβC/PLIN2 associations. Augmenting cellular LSDs that affect PLIN2 associations with these proteins, oleic acid (OA) treatment inhibits Wnt-increased AGβC/PLIN2 associations and β-catenin T-cell factor signaling (β-CTS). Revealing that PLIN2 is a GSK3-associated protein, the study explored PLIN2-mediated effects on GSK3/GSK3 substrates. PLIN2 siRNA reduces inhibitory GSK3 levels and lithium chloride (LiCl)-upregulated β-catenin or CCAAT/enhancer binding protein α (c/EBPα) expression. OA treatment decreases LiCl-increased c/EBPα via PLIN2-c/EBPα dissociation. In addition to PLIN2 overexpression increasing β-CTS, PLIN2 depletion or overexpression drops or adds expression of GSK3 substrates, such as β-catenin, c/EBPα,c-Myc, cyclin D1, and insulin receptor substrate 1, and cell growth/survival. PLIN2 N or C terminus overexpression that is associated with higher levels of the substrates suggests that those substrates bind to specific regions of PLIN2. Mimicking the possible high lipid concentrations in cells in the human body under conditions of hyperlipidemia/obesity, OA-treated cells gain or reduce GSK3 substrate expression in parallel with a decrease (a Wnt-like effect) or increase in GSK3 activity, likely regulated by GSK3/PLIN2/GSK3 substrate associations.

Decreased glycogen synthase kinase-3 levels and activity contribute to Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by brain atrophy particularly in striatum leading to personality changes, chorea and dementia. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase in the crossroad of many signaling pathways that is highly pleiotropic as it phosphorylates more than hundred substrates including structural, metabolic, and signaling proteins. Increased GSK-3 activity is believed to contribute to the pathogenesis of neurodegenerative diseases like Alzheimer's disease and GSK-3 inhibitors have been postulated as therapeutic agents for neurodegeneration. Regarding HD, GSK-3 inhibitors have shown beneficial effects in cell and invertebrate animal models but no evident efficacy in mouse models. Intriguingly, those studies were performed without interrogating GSK-3 level and activity in HD brain. Here we aim to explore the level and also the enzymatic activity of GSK-3 in the striatum and other less affected brain regions of HD patients and of the R6/1 mouse model to then elucidate the possible contribution of its alteration to HD pathogenesis by genetic manipulation in mice. We report a dramatic decrease in GSK-3 levels and activity in striatum and cortex of HD patients with similar results in the mouse model. Correction of the GSK-3 deficit in HD mice, by combining with transgenic mice with conditional GSK-3 expression, resulted in amelioration of their brain atrophy and behavioral motor and learning deficits. Thus, our results demonstrate that decreased brain GSK-3 contributes to HD neurological phenotype and open new therapeutic opportunities based on increasing GSK-3 activity or attenuating the harmful consequences of its decrease.