Role Of GSK-3β Research Articles

GSK-3β Contributes to Parkinsonian Dopaminergic Neuron Death: Evidence From Conditional Knockout Mice and Tideglusib.

Glycogen synthase kinase-3 (GSK-3) dysregulation has been implicated in nigral dopaminergic neurodegeneration, one of the main pathological features of Parkinson’s disease (PD). The two isoforms, GSK-3α and GSK-3β, have both been suggested to play a detrimental role in neuronal death. To date, several studies have focused on the role of GSK-3β on PD pathogenesis, while the role of GSK-3α has been largely overlooked. Here, we report in situ observations that both GSK-3α and GSK-3β are dephosphorylated at a negatively acting regulatory serine, indicating kinase activation, selectively in nigral dopaminergic neurons following exposure of mice to 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP). To identify whether GSK-3α and GSK-3β display functional redundancy in regulating parkinsonian dopaminergic cell death, we analysed dopaminergic neuron-specific Gsk3a null (Gsk3aΔDat) and Gsk3b null (Gsk3bΔDat) mice, respectively. We found that Gsk3bΔDat, but not Gsk3aΔDat, showed significant resistance to MPTP insult, revealing non-redundancy of GSK-3α and GSK-3β in PD pathogenesis. In addition, we tested the neuroprotective effect of tideglusib, the most clinically advanced inhibitor of GSK-3, in the MPTP model of PD. Administration of higher doses (200 mg/kg and 500 mg/kg) of tideglusib exhibited significant neuroprotection, whereas 50 mg/kg tideglusib failed to prevent dopaminergic neurodegeneration from MPTP toxicity. Administration of 200 mg/kg tideglusib improved motor symptoms of MPTP-treated mice. Together, these data demonstrate GSK-3β and not GSK-3α is critical for parkinsonian neurodegeneration. Our data support the view that GSK-3β acts as a potential therapeutic target in PD and tideglusib would be a candidate drug for PD neuroprotective therapy.

O-GlcNAc impairs endothelial function in uterine arteries from virgin but not pregnant rats: The role of GSK3β

Increased O-Linked β-N-acetylglucosamine (O-GlcNAc) is observed in several pathologies, and unbalanced O-GlcNAcylation levels favor endothelial dysfunction. Whether augmented O-GlcNAc impacts the uterine artery (UA) function and how it affects the UA during pregnancy remains to be elucidated. We hypothesized that glucosamine treatment increases O-GlcNAc, leading to uterine artery dysfunction and this effect is prevented by pregnancy. Pregnant (P) and non-pregnant (NP) Wistar rats were treated with glucosamine (300 mg/kg; i.p.) for 21 days. Concentration response-curves (CRC) to acetylcholine (in the presence or absence of L-NAME) and sodium nitroprusside were performed in UAs. In NP rats, glucosamine treatment increased O-GlcNAc expression in UAs accompanied by decreased endothelium-dependent relaxation, which was abolished by L-NAME. Endothelial nitric oxide synthase (eNOS) activity and total Akt expression were decreased by glucosamine-treatment in NP rats.Further, NP rats treated with glucosamine displayed increased glycogen synthase kinase 3 beta (GSK3β) activation and O-GlcNAc-transferase (OGT) expression in the UA. P rats treated with glucosamine displayed decreased O-GlcNAc in UAs and it was accompanied by improved relaxation to acetylcholine, whereas eNOS and GSK3β activity and total Akt and OGT expression were unchanged. Sodium nitroprusside-induced relaxation was not changed in all groups, indicating that glucosamine treatment led to endothelial dysfunction in NP rats. The underlying mechanism is, at least in part, dependent on Akt/GSK3β/OGT modulation. We speculate that during pregnancy, hormonal alterations play a protective role in preventing O-GlcNAcylation-induced endothelial dysfunction in the UAs.

Role of GSK3β and the A‐Type Potassium Channel in the Behavioral Phenotype of a Mouse Model of Parkinson’s Disease

Degeneration of dopaminergic (DA) neurons is a hallmark of Parkinson’s disease (PD). The mechanism involved in the site‐specific dysfunction of DA in the substantia nigra pars compacta (SNpc) but not in the ventral tegmental area (VTA) is not well understood. Glycogen Synthase Kinase 3 beta (GSK3β) activity has been shown to be increased in mouse models of PD and implicated in the human disease. Here we use the rotarod test to evaluate locomotion and balance, the open field test to evaluate anxiety and locomotion and whole cell current studies of DA neurons in brain slices of the midbrain to test the hypothesis that increased GSK3β activity in a mouse model for rapidly progressive PD inhibits A‐Type K+ channel activity specifically in the SNpc compared with the VTA, resulting in motor and non‐motor dysfunction in these mice. Using mice that overexpress A53Tα‐synuclein (A53T) under the pituitary specific PITX3 promoter as an animal model for PD, which developed gait disturbances as early as 7 weeks of age, we determined that A53T mice demonstrated decreased rotarod time compared with controls (134.4±27.0 sec (n=15) vs 287.1±13.0 sec (n=30); p < 0.001). Furthermore, treatment for 3 weeks with a GSK3β inhibitor, TWS‐119, resulted in an increase in rotarod time after 1 week (2.4±0.55 fold (n=5); p = 0.043), which was stable for up to 3 weeks. Furthermore, the open field test demonstrated that A53T mice spent more time in the periphery and traveled less in the center as compared to control mice (78.6±16.3 cm (n=17) vs 177.8±24.0 cm (n=30); p=0.006), and treatment with TWS‐119 led to a decrease in time spent in the periphery and an increase in distance traveled in the center as compared to control, indicating that GSK3β inhibition improved motor and non‐motor functions in this model. Whole‐cell patch‐clamp recordings of DA neurons from brain slices of A53T mice demonstrated a nearly 2‐fold increase in the incidence of spontaneous firing of DA neurons in the SNpc in response to current injection compared to WT (34.8±5.5 pA (n=14) vs 16±5.4 pA (n=6); p=0.04), while firing of DA neurons in slices from TWS‐119 treated A53T mice was not significantly different from WT. There was no difference in firing frequency in DA neurons in the VTA from brain slices from WT and A53T mice. Measurements of A‐Type K+ currents in SNpc of brain slices of A53T mice demonstrated a marked decrease in current density compared to WT (87.4±14.0pA (n=8) vs 164.7±32.4pA (n=19); p<0.001), while A‐Type currents in brain slices from TWS‐119 treated mice were not significantly different from control. Moreover, we identified a putative target site of GSK3β in the Kv4.2 structural subunits of the A‐Type K+ channel. These data support the conclusion that hyperactivity of GSK3β might play a role in the development of a PD behavioral phenotype in the PITX3/A53T mouse model for PD by mediating an increase in excitability of DA neurons via inhibition of the A‐type K channel repolarizing current specifically in DA neurons of the SNpc. This suggests the A‐Type K channel and its subunits as potential therapeutic targets in PD.Support or Funding InformationFunded by The Michael J. Fox Foundation

TDZD-8 alleviates delayed neurological sequelae following acute carbon monoxide poisoning involving tau protein phosphorylation

Objective: Acute carbon monoxide （CO）poisoning can cause delayed neurological sequelae (DNS). Glycogen synthase kinase 3β (GSK-3β) /Tau protein pathway is reported to play a key role in neurological abnormalities. In the present study, we aimed to determine the role of GSK-3β/Tau in DNS following acute CO poisoning.Methods: 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a specific non-competitive inhibitor of GSK-3β, was used to inhibit GSK-3β. Twenty-four male Sprague-Dawley rats were randomly assigned to the three groups: Control group, CO group and CO-TDZD-8 group. Rats breathed 1000 ppm CO for 40 minutes and then 3000 ppm for up to 20 minutes until they lost consciousness. TDZD-8 (1 mg/kg) was administered intravenously three times after the end of CO exposure at 0, 24, 48 hours late. Learning and memory abilities were observed using the Morris Water Maze (MWM). Brain histological changes were evaluated by hematoxylin-eosin staining. Moreover, the expression levels of Tau and GSK-3β were detected after acute carbon monoxide poisoning.Results: TDZD-8 significantly attenuated the learning and memory dysfunction induced by acute CO poisoning, ameliorated the histology structure of damaged neural cells in cortex and hippocampus CA1 area. TDZD-8 clearly decreased p-Tau expression, reversed the reduction of p-GSK-3β induced by acute CO poisoning.Conclusions: The therapeutic effect of TDZD-8 in alleviating DNS caused by acute CO poisoning is related to the inactivation of Tau by intensifying the level of GSK-3β phosphorylation.

The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma.

Rationale: Glycogen synthase kinase-3β (GSK-3β) plays key roles in metabolism and many cellular processes. It was recently demonstrated that overexpression of GSK-3β can confer tumor growth. However, the expression and function of GSK-3β in hepatocellular carcinoma (HCC) remain largely unexplored. This study is aimed at investigating the role and therapeutic target value of GSK-3β in HCC.Methods: We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we studied whether GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Co-immunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to explore the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments.Results: We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Overexpression of GSK-3β confers HCC cell colony formation and xenograft tumor growth. Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ.Conclusions: Our findings demonstrate that GSK-3β is a disruptor of retinoid signalling and a new resistant factor of sorafenib in HCC. Targeting GSK-3β may be a promising strategy for HCC treatment in clinic.

Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection.

BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated.METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy.RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β.CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Glycogen synthase kinase 3β hyperactivity in urinary exfoliated cells predicts progression of diabetic kidney disease

Burgeoning evidence points to glycogen synthase kinase (GSK)3β as a key player in diverse kidney diseases. However, as a pivotal transducer of the insulin signaling pathway, the role of GSK3β in diabetic kidney disease remains uncertain. In db/db mice, renal expression of total and activated GSK3β was increasingly elevated. This preceded the development of diabetic kidney disease, and correlated with the progression of signs of diabetic kidney injury, including albuminuria and extracellular matrix accumulation in glomeruli and tubulointerstitia. Invitro, exposure of glomerular podocytes, mesangial cells, and renal tubular cells to a diabetic milieu induced GSK3β overexpression and hyperactivity, which seem essential and sufficient for eliciting diabetic cellular damages in kidney cells, because the cytopathic effect of the diabetic milieu was mitigated by GSK3β knockdown, but was mimicked by ectopic expression of constitutively active GSK3β even in the normal milieu. In consistency, kidney biopsy specimens procured from patients with varying stages of diabetic nephropathy revealed an amplified expression of total and activated GSK3β in glomeruli and renal tubules, associated with the severity of diabetic nephropathy. Moreover, in retrospective cohorts of type 2 diabetic patients that were followed for over five years, the relative activity of GSK3β in banked urinary exfoliated cells represented an independent risk factor for development or progression of renal impairment. Furthermore, receiver operating characteristic curve analysis demonstrated that GSK3β activity in urinary exfoliated cells provided much better power than albuminuria in discriminating diabetic patients with progressive renal impairment from those with stable kidney function. Thus, renal expression and activity of GSK3β are amplified in experimental and clinical diabetic nephropathy. Hence, GSK3β in urinary exfoliated cells may serve as a novel biomarker for predicting diabetic kidney disease progression.

Abstract 616: Glycogen Synthase Kinase 3b Localizes to and Modulates Sarcomere Function in Health and Disease

Ischemic damage to a region of myocardium renders it weaker than the surrounding tissue, leading to contractile heterogeneity. This heterogeneity causes abnormal stress and strain patterns, further reducing systolic function. We hypothesized glycogen synthase kinase 3β (GSK-3β) may be a therapeutic target for improving function in patients with ischemic damage, as we previously discovered its activity is reduced by abnormal mechanical stress and it can phosphorylate myofilament proteins. We measured GSK-3β levels in LV whole tissue lysate from patients with ischemic cardiomyopathy (ICM) and non-failing rejected donor hearts (NF), but detected no differences. However, we found GSK-3β present at the myofilament, and ICM patients had a ~70% reduction in this localized pool compared to NF controls. Furthermore, GSK-3β phosphorylated at Y216 (pY216), an understudied site, was highly enriched in the myofilament. We used immunofluorescence, co-immunoprecipitation, and adenoviral constructs of Y216 phospho-mimetic or -blocked, in human, mouse, or neonatal rat ventricular myocytes to confirm Y216 phosphorylation results in GSK-3β binding to the myofilament. pY residues can dock to SH2 domains. The only SH2-containing protein that associates with the myofilament is tensin-1 (discovered by us in the myofilament by mass spectrometry). Here we further found tensin-1 localizes to the cardiac z-disk. GSK-3β and tensin-1 co-IP together in myocytes and in HEK cells (in the absence of the myofilament), but this interaction is reduced if Y216 phosphorylation is blocked. To identify the functional consequences of this interaction, we utilized an inducible myocyte specific GSK-3β knock-out mouse. Skinned myocytes from GSK-3β KO mice had significantly depressed calcium sensitivity compared to WT mice. Furthermore, treating skinned myocytes isolated from ICM patients with GSK-3β significantly increased calcium sensitivity, but had no effect in NF cells. These findings identify a novel functional role for GSK-3β in the ischemic human heart. GSK-3β is a promiscuous kinase, but we have discovered a specific regulatory mechanism (Y216 phosphorylation) that could allow precise therapeutic intervention to improve contractile function in ICM patients.

The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma

Background: Glycogen synthase kinase-3β (GSK-3β) is recently demonstrated to be a tumor promoter and a potential drug target in several tumors. However, the implication and mechanism of GSK-3β in hepatocellular carcinoma (HCC) remain unexplored. Methods: We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we investigated where GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Coimmunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to study the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments. Findings: We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition: from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ. Interpretation: Our findings suggest that overexpression of GSK-3β may confer HCC growth through interfering retinoid signalling. Targeting GSK3β can be a promising strategy for designing improved treatment of sorafenib in HCC. Funding Statement: This work is supported by grants from Natural Science Foundation of China (No. 81673467, 31471273, 31461163002/RGC N_HKU 740/14), the 10th Singapore-China Joint Research Program (S2014GR0448), Natural Science Foundation of Fujian Province (2019I0002) and Leading Talents in Scientific and Technological Innovation, Double Hundred Talents Program of Fujian Province. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All the use of human samples and study protocols were approved by the Hospital Ethics Committee. All patients signed an informed consent form in prior to sample collection.

OCT4B-190 protects against ischemic stroke by modulating GSK-3β/HDAC6

OCT4 is a key regulator in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Human OCT4 gene has three mRNA isoforms, termed OCT4A, OCT4B and OCT4B1. The 190-amino-acid protein isoform (OCT4B-190) is one of the major products of OCT4B mRNA, the biological function of which is still not well defined. Recent evidence suggests that OCT4B-190 may function in the cellular stress response. The glycogen synthase kinase-3β (GSK-3β) and histone deacetylase 6 (HDAC6) are also key stress modulators that play critical roles in the ischemic cascades of stroke. Hence, we here further investigated the effects of OCT4B-190 in the experimental stroke, and explored the underlying roles of GSK-3β and HDAC6. We found that OCT4B-190 overexpression enhanced neuronal viability at 24 h after oxygen-glucose deprivation (OGD) treatment. Moreover, in male C57BL/6 mice subjected to transit middle cerebral artery occlusion (MCAO), OCT4B-190 overexpression reduced infarct volume and improved neurological function after stroke. Notably, we found spatio-temporal alterations of GSK-3β and HDAC6 in the ischemic cortex and striatum, which were affected by adenovirus-mediated OCT4B-190 overexpression. OCT4B-190 demonstrated similar impacts on neuronal cultures in vitro, downregulating OGD-induced GSK-3β activity and HDAC6 expression. In addition, we found that GSK-3β and HDAC6 were co-expressed in the cytoplasm of neurons, and OCT4B-190 had an effect on interactions between GSK-3β and HDAC6 in neuronal cultures subjected to OGD treatment. These findings suggest that OCT4B-190 exerts neuroprotection in the experimental stroke potentially by regulating actions of GSK-3β and HDAC6 simultaneously, which may be an attractive therapeutic strategy for ischemic stroke.

GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2-4 cell stage in Lymnaea stagnalis.

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3β, β-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3β and β-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3β inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of β-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3β in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

Molecular cloning and transcriptional and functional analysis of glycogen synthase kinase-3β in Haemaphysalis longicornis (Acari, Ixodidae).

Glycogen synthase kinase 3 (GSK-3), which belongs to the serine/threonine kinase family, regulates glycogen metabolism, Wnt signaling, hormonal regulation, and embryonic development in many eukaryotes. Here, we cloned a complete open reading frame (ORF) of glycogen synthase kinase 3β (GSK-3β) from Haemaphysalis longicornis and characterized its transcriptional and functional status. The ORF of GSK-3β possesses 1242 nucleotides encoding a mature protein of 413 amino acid residues. GSK-3β nucleotide and protein sequences are highly conserved among different vertebrate and invertebrate animals, with identity between 47.8–100% and 63.2–88.7%, respectively. Sequence comparison showed one signature domain between the residues of 51 and 335 amino acids, which was identified as a protein kinase (serine/threonine). RT-PCR showed GSK-3β mRNA present in all developmental stages of H. longicornis. Interestingly, a higher transcript level was observed in nymph and 7-day-old eggs compared with others by real-time PCR, indicating a role of GSK-3β in the early stages of life. The functional status of GSK-3β was characterized by RNA interference (RNAi) and caused significant (p < 0.05) reduction in feeding and reproduction, as well as an abnormality in eggs and hatching. Taken together, our results suggest that GSK-3β may be an important candidate for a multiple antigen vaccine for controlling the tick population.

The Possible Interactions and Therapeutic Roles of Lithium Chloride and Midkine on Cancer Treatment.

Midkine (MK) is a heparin-binding anti-apoptotic growth factor or cytokine also known as neurite growth-promoting factor 2 (NEGF2). It is developmentally an important retinoic acid-responsive gene product strongly induced during the mid-gestation stage. Midkine promotes different cellular events such as cell growth, differentiation, survival, gene expression, and drug resistence. Midkine, the phosphatidylinositol 3-kinases (PI3-kinase, PI3K) and glycogen synthase kinase-3 beta (GSK-3β) inhibitors together with lithium chloride may be a very effective treatment modality, especially in tumors with high expression of these two molecules. PI3 kinase and GSK-3β, both serine threonine kinases located in the center of the signaling network, are very important regulator molecules for cell survival or death. Lithium chloride (LiCl), with its newly discovered antineoplastic effect and cytotoxicity potentiation, has become a promising agent in the application of new combination treatments. Although the LiCl mechanism of action is still not fully understood, previous studies have shown that LiCl is an inhibitör of the inositol monophosphatase (IMPase) and GSK-3β. GSK-3β, is a serine-threonine protein kinase involved in cell proliferation, differentiation, survival, apotosis, and tumorogenesis. The role of GSK-3β in tumorigenesis and cancer remains controversial. It may have a function as a tumor suppressor for certain types of tumors, but it promotes cell growth and development in other tumor types.

Antidepressant effects of creatine on amyloid β1–40-treated mice: The role of GSK-3β/Nrf2 pathway

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction and neuronal lost in specific brain areas including hippocampus, resulting in memory/learning deficits and cognitive impairments. In addition, non-cognitive symptoms are reported in AD patients, such as anxiety, apathy and depressed mood. The current antidepressant drugs present reduced efficacy to improve depressive symptoms in AD patients. Here, we investigated the ability of creatine, a compound with neuroprotective and antidepressant properties, to counteract amyloid β1–40 peptide-induced depressive-like behavior in mice. Moreover, we addressed the participation of the intracellular signaling pathway mediated by glycogen synthase kinase-3β (GSK-3β)/nuclear factor erythroid-2-related factor 2 (Nrf2) in the creatine effects. Aß1–40 administration (400 pmol/mouse, i.c.v.) increased the immobility time in the tail suspension test and decreased the grooming time and increased latency to grooming in the splash test, indicative of depressive-like behavior. These impairments were attenuated by creatine (0.01 and 10 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o., positive control). No significant alterations on locomotor performance were observed in the open field. Aß1–40 administration did not alter hippocampal phospho-GSK-3β (Ser9)/total GSK-3β, total GSK-3β and heme oxygenase-1 (HO-1) immunocontents. However, Aß1–40-infused mice treated with creatine (0.01 mg/kg) presented increased phosphorylation of GSK-3β(Ser9) and HO-1 immunocontent in the hippocampus. Fluoxetine per se increased GSK-3β(Ser9) phosphorylation, but did not alter HO-1 levels. In addition, Aß1–40 administration increased hippocampal glutathione (GSH) levels as well as glutathione reductase (GR) and thioredoxin reductase (TrxR) activities, and these effects were abolished by creatine and fluoxetine. This study provides the first evidence of the antidepressive-like effects of creatine in Aß1–40-treated mice, which were accompanied by hippocampal inhibition of GSK-3β and modulation of antioxidant defenses. These findings indicate the potential of creatine for the treatment of depression associated with AD.

GSK3β Regulates Brain Energy Metabolism

SUMMARYGSK3β is a serine threonine kinase implicated in the progression of Alzheimer’s disease. Although the role of GSK3β in growth and pathology has been extensively studied, little is known about the metabolic consequences of GSK3β manipulation, particularly in the brain. Here, we show that GSK3β regulates mitochondrial energy metabolism in human H4 neuroglioma cells and rat PC12-derived neuronal cells and that inhibition of GSK3β in mice in vivo alters metabolism in the hippocampus in a region-specific manner. We demonstrate that GSK3β inhibition increases mitochondrial respiration and membrane potential and alters NAD(P)H metabolism. These metabolic effects are associated with increased PGC-1α protein stabilization, enhanced nuclear localization, and increased transcriptional co-activation. In mice treated with the GSK3β inhibitor lithium carbonate, changes in hippocampal energy metabolism are linked to increased PGC-1α. These data highlight a metabolic role for brain GSK3β and suggest that the GSK3β/PGC-1α axis may be important in neuronal metabolic integrity.

Role of GSK-3β in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats: evaluation using RNA interference adenovirus infection method

Objective To evaluate the role of glycogen synthase kinase 3 beta (GSK-3β) in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats using RNA interference (RNAi) adenovirus infection method. Methods H9C2 cells were transferred into 96-well cell plates at a density of 1×105 cells/ml after culture and then divided into 8 groups (n=10 each) using a random number table: control group (group C), bupivacaine group (group B), lipid emulsion group (group LE), bupivacaine plus lipid emulsion group (group B+ LE), control plus GSK-3βRNAi adenovirus (GSK-3βi) group (group C+ GSK-3βi), bupivacaine plus GSK-3βi group (group B+ GSK-3βi), lipid emulsion plus GSK-3βi group (group LE+ GSK-3βi) and bupivacaine plus lipid emulsion plus GSK-3βi group (group B+ LE+ GSK-3βi). In B, LE and B+ LE groups, the cells were incubated with culture medium containing 1 mmol/L bupivacaine, 1% lipid emulsion and 1 mmol/L bupivacaine plus 1% lipid emulsion, respectively.In C+ GSK-3βi, B+ GSK-3βi, LE+ GSK-3βi and B+ LE+ GSK-3βi groups, the cells were incubated with the drugs mentioned above on 2nd day after being infected by adenovirus.At 24 h after incubation with drugs, the expression of Bax and Bcl-2 was determined by Western blot, and the apoptosis rate was calculated using DAPI staining. Results Compared with group C, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated, and the apoptosis rate was increased in group B (P<0.05). Compared with group B, the expression of Bax was significantly down-regulated, the expression of Bcl-2 was up-regulated, and the apoptosis rate was decreased in group B+ LE (P<0.05). Compared with group B+ LE, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated and the apoptosis rate was increased in group B+ LE+ GSK-3βi (P<0.05). Conclusion The mechanism by which lipid emulsion inhibits bupivacaine-induced apoptosis in cardiomyocytes of rats is associated with GSK-3β. Key words: Fat emulsions, intravenous; Bupivacaine; Apoptosis; Myocytes, cardiac

Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3β that plays different but often overlapping functions. Although the role of GSK3β in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated–inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3β at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3β with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

Inactivation of glycogen synthase kinase-3β (GSK-3β) enhances skeletal muscle oxidative metabolism

BackgroundAberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. Evidence in non-muscle cells suggests that glycogen synthase kinase-3β (GSK-3β) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3β in the regulation of skeletal muscle oxidative metabolism is unknown. AimsWe hypothesized that inactivation of GSK-3β stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3β inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism. MethodsGSK-3β was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3β knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated. ResultsIn vitro, knock-down of GSK-3β increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3β increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3β KO animals were protected against unloading-induced decrements in expression levels of these constituents. ConclusionInactivation of GSK-3β up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3β KO protects against inactivity-induced reductions in muscle metabolic gene expression.

Role of GSK-3β in Regulation of Canonical Wnt/β-catenin Signaling and PI3-K/Akt Oncogenic Pathway in Colon Cancer

ABSTRACTNon-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents because of their ability in blocking cellular proliferation, and thereby tumor development, and also by promoting apoptosis. GSK-3β, a serine threonine kinase and a negative regulator of the oncogenic Wnt/β-catenin signaling pathway, plays a critical role in the regulation of oncogenesis. Celecoxib and etoricoxib, the two cyclooxygenase-2 (COX-2) selective NSAIDs, and Diclofenac, a preferential COX-2 inhibitory NSAID, had shown uniformly the chemopreventive and anti-neoplastic effects in the early stage of colon cancer by promoting apoptosis as well as an over-expression of GSK-3β while down-regulating the PI3-K/Akt oncogenic pathway.

Abstract 4120: Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients

Abstract Background: PI3K/Akt/mTORC1 signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated that glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. Herein, we aimed to investigate how the mTORC1 downstream substrate 4EBP1 is directly regulated by GSK-3β leading to acquisition of clinical chemoresistance to mTORC1 inhibitor (mTORi). Methods: The expression and phosphorylation of molecules in the subcellular pathways were examined by immunoblotting in surgically resected RCC tissues and human RCC cell lines including ACHN. Rapamycin-resistant ACHN cells (ACHN/RR) were generated by chronic exposure to an mTORi, rapamycin. Cell viability was investigated by MTS assay. The direct role of GSK-3β in regulating 4EBP1 was evaluated by an in vitro kinase assay. Pharmacological inhibition of GSK-3β, PI3K/Akt/mTORC1 pathway, and mTORC1 was achieved by treatment with AR-A014418, LY294002, and rapamycin, respectively. The effects of drug combination were determined using CompuSyn software. GSK-3β expression was evaluated in pathological RCC tissues obtained by nephrectomy from patients with metastatic RCC (mRCC), nine of whom were treated with mTORi as systemic therapies. GSK-3β expression was immunohistochemically graded as high and low expression using a semi-quantitative method based on positive staining intensity. Survival intervals were evaluated using the Kaplan-Meier method and log-rank test. Significance level was set as 0.05 in statistical analysis using a freeware R. Results: Inhibition of PI3K/Akt/mTORC1 pathway and mTORi treatment sufficiently decreased pS6RP, but only moderately decreased p4EBP1. In contrast to the effect of rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 in ACHN and ACHN/RR. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1 at Thr37, Thr46, Thr70 and Ser65, and these phosphorylations were blocked by GSK-3 inhibitor. A combination of AR- A014418 and rapamycin produced an additive effect at lower concentrations, and were antagonistic at higher concentrations. Immunoblotting demonstrated that 4EBP1 and p4EBP1 expression in RCC tissues was positively correlated with GSK-3β expression. Overall survival was significantly short in patients with high GSK-3β expression, compared with those having low expression (n = 25 and 14, respectively, p = 0.04). In addition, high GSK-3β expression in tumors tended to shorten progression-free intervals in mRCC patients treated with mTORi. Conclusions: High GSK-3ββ expression and direct GSK-3β/4EBP1 pathway can be a key mechanism for RCC acquiring clinical chemoresistance to mTORCi. Activation of the direct pathway might be predictive of poor prognosis of mRCC patients. Citation Format: Hiromi Ito, Sei Naito, Osamu Ichiyanagi, Hidenori Kanno, Yuta Kurota, Atushi Yamagishi, Vladimir Bilim, Yoshihiko Tomita, Tomoyuki Kato, Akira Nagaoka, Norihiko Tsuchiya. Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4120. doi:10.1158/1538-7445.AM2017-4120