Inhibition Of GSK-3beta Research Articles

GSK-3beta Inhibitor Induces Expression of Nrf2/TrxR2 Signaling Pathway to Protect against Renal Ischemia/Reperfusion Injury in Diabetic Rats

Background/Aims: Diabetes mellitus (DM) can lead to renal damage and dysfunction, and exacerbate renal ischemia/reperfusion injury (RI/RI). The aim of this study was to investigate the protective effect of GSK-3β inhibitor TDZD-8 against RI/RI through Nrf2/TrxR2 signaling pathway in a rat DM model. Methods: A DM rat model was established by a single injection of streptozocin. Diabetic rats were pretreated with TDZD-8 (1 mg/kg bw) or TDZD-8+auranofin (10 nmol/L, 5ml/kg bw), and then subjected to 45-min ischemia and 24-h reperfusion. Rats were equally randomized into four groups: a Sham-operated group, a RI/RI group, a TDZD-8 group, and a TDZD-8+auranofin group. Serum levels of BUN and Scr were measured. SOD activity, MDA content, and Nrf2, TrxR2 and caspase-3 expressions in rat kidney tissues were determined. Results: Renal function was improved, oxidative stress and cell apoptosis were reduced, and the expression of Nrf2 and TrxR2 was up-regulated in TDZD-8 treated rats as compared with those in auranofin treated rats. Conclusion: TDZD-8 may exert its protective effect against RI/RI by regulating the Nrf2/TrxR2 signaling pathway in the kidney tissue in DM.

Aeroallergen Der p 2 induces apoptosis of bronchial epithelial BEAS-2B cells via activation of both intrinsic and extrinsic pathway.

BackgroundExcessive apoptosis of airway epithelium is reported to induce airway remodeling and inhibited airway epithelium repair is highly associated with development of asthma and chronic obstructive pulmonary disease. Der p 2 is a major allergen derived from Dermatophagoides pteronyssinus and commonly causes airway hypersensitiveness and asthma; however, the connection between Der p 2 and epithelial apoptosis remains unclear. This study was aimed to explore whether Der p 2 induces apoptosis of airway epithelial cells and the underlying mechanisms.ResultsOur results showed that recombinant Der p 2 (rDP2) inhibited cell growth and induced apoptosis of human bronchial epithelial cell BEAS-2B. Further investigation revealed that rDP2 increased intracellular reactive oxygen species, level of cytosolic cytochrome c and cleavage of caspase-9 and caspase-3. rDP2 also induced activation of p38 mitogen-activated protein kinase (P38) and c-Jun N-terminal kinase (JNK), and triggered proapoptotic signals including decrease of Bcl-2, increase of Bax and Bak, and upregulation of Fas and Fas ligand. In parallel, rDP2 inhibited glycogen synthase kinase 3beta and consequently enhanced degradation of cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP). Involvement of toll-like receptor (TLR)2 in rDP2-induced apoptosis was also demonstrated using specific small inhibitory RNA.ConclusionsOur findings indicate that rDP2 suppresses cell growth and trigger apoptosis of BEAS-2B cells, which may attribute to induction of both intrinsic and extrinsic pathway via TLR2 and P38/JNK signaling and c-FLIP degradation. It suggests that Der p 2 may aggravate respiratory disorders through enhancement of apoptosis and the consequent airway injury.

Abstract B25: R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature

Abstract Formation of endothelial lumen is fundamental to angiogenesis and generation of functional vasculature. Tumors develop a large number of angiogenic blood vessels. However, a significant fraction of these vessels are poorly functional due to the impaired vessel maturation such as the improper formation of endothelial lumen. R-Ras is a small GTPase of the Ras family, which inhibits vessel sprouting and branching activities and redirects the vessel sprouts from these activities to the vessel maturation process. Through this effect, R-Ras normalizes pathologically regenerating blood vessels. Here we show that R-Ras regulates formation of endothelial lumen in the developing vessel sprouts. R-Ras deficient mice develop a large number of poorly perfused blood vessels in response to tissue ischemia. Notably, many of these vessels lack lumen assessed by the lack of PODXL staining of the endothelium. In 3D in vitro angiogenesis studies in fibrin gels, upregulation of R-Ras dramatically enhances formation of lumens in the endothelial sprouts. On the other hand, knockdown of the endogenous R-Ras abrogates lumen formation and endothelial cell differentiation into tubular structures resulting in abnormal extension of endothelial cells with numerous membrane protrusions. The stabilization of microtubules in endothelial cells is critical to the formation of endothelial lumen. Upregulation of R-Ras signaling in endothelial cells results in the formation of prominent microtubule cytoskeleton network that extends to the membrane periphery in 2D culture. This phenomenon is accompanied by the significant spreading of endothelial cells. We found that R-Ras stabilizes microtubules by increasing acetylation of alpha-tubulin. This effect of R-Ras is mediated by the PI3-kinase-Akt1/2 pathway, which phosphorylates GSK3-beta and inhibits the inhibitory activity of GSK3-beta on tubulin acetylation. GSK3-beta inhibition partially rescues tubulogenesis of R-Ras-silenced endothelial cells. These findings demonstrate a novel Akt-dependent mechanism of endothelial lumen formation and blood vessel maturation. The endothelial-specific upregulation of R-Ras in vivo leads to improved blood perfusion and normalization of VEGF-induced angiogenic vessels developing in Matrigel implants. The enhanced endothelial lumen formation and resulting improvement of vessel perfusion would significantly increase the delivery of chemotherapeutic drugs to tumors, which is otherwise hampered by the abnormal, poorly perfused vasculature. This R-Ras-dependent mechanism of endothelial lumen formation should be further exploited to potentially develop a strategy for improving drug delivery and enhancing treatment efficacy via normalization of the tumor vasculature. Citation Format: Fangfei Li, Junko Sawada, Masanobu Komatsu. R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B25.

Inhibition of GSK3-beta rescues hippocampal development in a knockout mouse model of CDKL5 encephalopathy

Inhibition of GSK3-beta rescues hippocampal development in a knockout mouse model of CDKL5 encephalopathy

Abstract 1957: Negative regulation of Sox9 by glycogen synthase kinase 3 beta phosphorylation and SCFFbw7-dependent ubiquitination in cancer

Abstract Sox9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of transcription factors, with known functions in proliferation, epithelial-mesenchymal transition (EMT), stem cell maintenance and senescence. Sox9 protein levels are elevated in several different cancer types, including lung cancer and osteosarcoma. Furthermore, Sox9 overexpression is associated with poor survival in both cancer types. We recently reported that Sox9 is a direct transcriptional target of the Notch signaling pathway and mediates Notch1-induced EMT in lung cancer (Capaccione et al., Oncotarget, 2014). Fbxw7 (F-box and WD repeat domain-containing 7) is a member of the SCFFbw7E3 ubiquitin ligase complexthat targets several oncogenic proteins for degradation, including Notch1. We hypothesized that Sox9 is targeted for degradation by SCFFbw7. Knocking down or overexpressingFbxw7 significantly increasedor decreased, respectively, endogenous Sox9 protein levels in lung cancer and osteosarcoma cell lines. In addition, overexpression of Fbxw7 resulted in active degradation of ectopic Sox9 protein levels, when assessed in combination with cycloheximide, and did so in a dose dependent-manner.Furthermore, both Sox9 and FBXW7 were detected in immunoblots after the reciprocal protein was immunoprecipitated. Because substrate degradation by SCFFbw7 is typically phosphorylation-dependent, frequently by glycogen synthase kinase (GSK)3-beta, we examined whether modulation of GSK3-beta could affect FBXW7-induced Sox9 degradation. Treatment of cells with the GSK3-beta inhibitor, CHIR99021, completely abolished Sox9 degradation by ectopically overexpressed FBXW7, in a dose-dependent manner. We next identified four putative phosphorylation sites within potential FBXW7 degron motifs in Sox9.Mass spectrophotometric analysis confirmed these sites within Sox9 are indeed phosphorylated.In summary, we found that the E3 ubiquitin ligase complex (SCFFbw7) targets Sox9 for ubiquitin-mediated degradation in a GSK3-beta-mediated Sox9 phosphorylation-dependent manner. As a result, this negative regulation of Sox9 by GSK3-beta/FBXW7 could lead to inhibition of Sox9-mediated proliferation, EMT and stemness. This new regulatory mechanism of Sox9 could have diagnostic and therapeutic implications for lung cancer, osteosarcoma, as well as other cancers. Citation Format: Xuehui Hong, Wenyu Liu, Hiroyuki Inuzuka, Lianxin Liu, Sharon R. Pine. Negative regulation of Sox9 by glycogen synthase kinase 3 beta phosphorylation and SCFFbw7-dependent ubiquitination in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1957. doi:10.1158/1538-7445.AM2015-1957

Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2

Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion.

Lentiviral silencing of GSK-3β in adult dentate gyrus impairs contextual fear memory and synaptic plasticity.

Attempts have been made to use glycogen synthase kinase-3 beta (GSK3β) inhibitors for prophylactic treatment of neurocognitive conditions. However the use of lithium, a non-specific inhibitor of GSK3β results in mild cognitive impairment in humans. The effects of global GSK3β inhibition or knockout on learning and memory in healthy adult mice are also inconclusive. Our study aims to better understand the role of GSK3β in learning and memory through a more regionally, targeted approach, specifically performing lentiviral-mediated knockdown of GSK3β within the dentate gyrus (DG). DG-GSK3β-silenced mice showed impaired contextual fear memory retrieval. However, cue fear memory, spatial memory, locomotor activity and anxiety levels were similar to control. These GSK3β-silenced mice also showed increased induction and maintenance of DG long-term potentiation (DG-LTP) compared to control animals. Thus, this region-specific, targeted knockdown of GSK3β in the DG provides better understanding on the role of GSK3β in learning and memory.

Inhibition of GSK3beta leads to increased survival, proliferation and memory phenotype generation of GBM-specific CAR T cells (VAC5P.1125)

Abstract Application of chimeric antigen receptors (CAR), where an antibody or ligand-binding domain is fused with the zeta signaling chain of the T cell receptor, is a novel and alternative strategy to bypass the failure of cytotoxic immune response induction by most tumor cells. Although CARs have shown impressive preclinical efficacy, translational success in solid tumors like glioblastoma (GBM) is often restricted by the limited viability of the T cells either due to the absence of IL2 in the central nervous system and/or because of mandated chemotherapy. Although multiple doses of T cell transfer and systemic administration of IL2 are effective for short time periods, unregulated overdose of T cells and IL2 result in immune toxicity. Inhibition of glycogen synthase kinase 3 beta (GSK3β) in GBM-specific IL13CAR T cells increased IL2-independent survival and proliferation of the CAR-positive T cells upon activation with IL13Rα2-expressing GBM tumor cells or soluble target antigen, when compared to bystander CAR-negative T cells or untreated IL13CAR T cells. GSK3β-inhibited IL13CAR T cells also showed increased IL7R and CD45RO expression compared to the bystanders as well as untreated IL13CAR T cells indicating a possible development of memory population. Exposure to target antigen increased cytotoxic efficacy and tumor-killing ability specifically in GSK3β-inhibited IL13CAR T cells. These observations suggest an innovative advancement in anti-GBM CAR T cell-mediated immunotherapy.

Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). With the application of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), APL becomes one of best prognosis of leukemia. However, ATRA and ATO are not effective against all APLs. Therefore, a new strategy for APL treatment is necessary. Here, we investigated whether lithium chloride (LiCl), a drug used for the treatment of mental illness, could promote apoptosis in human leukemia NB4 cells. We observed that treatment with LiCl significantly accelerated apoptosis in NB4 cells and led to cell cycle arrest at G2/M phase. Moreover, LiCl significantly increased the level of Ser9-phosphorylated glycogen synthase kinase 3β(p-GSK-3β), and decreased the level of Akt1 protein in a dose-dependent manner. In addition, LiCl inhibition of c-Myc also enhanced cell death with a concomitant increase in β-catnin. Taken together, these findings demonstrated that LiCl promoted apoptosis in NB4 cells through the Akt signaling pathway and that G2/M phase arrest was induced by increase of p-GSK-3β(S9).

Tacrine, an oral acetylcholinesterase inhibitor, induced hepatic oxidative damage, which was blocked by liquiritigenin through GSK3-beta inhibition.

Although the cholinesterase inhibitor tacrine has been successfully used for the treatment of Alzheimer's disease, it is known to have hepatotoxic effects. Liquiritigenin (LQ), an active flavonoid in Glycyrrhizae radix, exerts protective effects against liver damage. This study investigated the toxic effect of tacrine on hepatocytes and the beneficial effect of LQ on tacrine intoxication in vivo and in vitro, and the underlying mechanism involved. In hepatocyte cell lines, tacrine induced cell death and oxidative stress, as indicated by decreases in cell viability and glutathione (GSH) contents, which were blocked by pretreatment with LQ. Fluorescent activated cell sorter (FACS) analysis revealed that LQ inhibited cellular H2O2 production and mitochondrial dysfunction induced by tacrine in HepG2 cells. Furthermore, LQ promoted inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β) and prevented decreases in GSK3β phosphorylation induced by tacrine. In rats treatment with tacrine at 30 mg/kg increased hepatic damage as assessed by blood biochemistry and histopathology. Administration of LQ (10 or 30 mg/kg/d, per os (p.o.)) or the hepatoprotective drug sylimarin (100 mg/kg/d) for 3 d inhibited elevations in alanine aminotransferase, aspartate aminotransferase, and histological changes induced by tacrine. These results show that LQ efficaciously protects the rat liver against tacrine-induced liver damage, and suggest that LQ is a therapeutic candidate for ameliorating the hepatotoxic effects of tacrine.

The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling.

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.

Molecular targeted therapy for osteosarcoma using glycogen synthase kinase-3 beta (GSK-3β) inhibitors.

10536 Background: Chemotherapy for osteosarcoma has improved the 5-year survival rate in osteosarcoma patients to 70%. However, development of new therapies for osteosarcoma is warranted, as chemot...

Down‐regulation of serum gonadotropins but not estrogen replacement improves cognition in aged‐ovariectomized 3xTg AD female mice

Development of Alzheimer's disease (AD) has been linked to the de-regulation of estrogen and gonadotropins such as luteinizing hormone (LH). In this study, we found increases in AD pathology in the hippocampi of aged female 3xTg AD mice after ovariectomy that were unable to be reduced by estrogen therapy or down-regulation of serum LH levels. Despite the lack of effect of these treatments on AD pathology, down-regulation of serum LH but not estrogen improved factors associated with neuronal plasticity such as spatial memory, inhibition of glycogen synthase kinase-3 beta, expression of beta-catenin, and brain-derived neurotrophic factor transcription. Contrasting previous studies in younger mice, estrogen replacement was not able to rescue behavioral deficits, reduced glycogen synthase kinase-3 beta inhibition and increased hippocampal phosphorylation of tau. Of critical importance, serum LH was negatively correlated with brain LH in regions associated with spatial memory, and increases in brain LH correlated with cognitive improvement. This paralleled changes in human female AD brains which showed a significant reduction in brain LH mRNA compared to healthy age- and PMI-matched controls. Taken together, these findings should promote further research into the LH-dependent mechanisms associated with AD cognitive deficits as well as the effects of estrogen within the aged brain. In the aged triple transgenic Alzheimer's disease (AD) mouse model (3xAD-Tg), estrogen replacement after ovariectomy does not improve cognitive function, increases phosphorylated Tau levels and decreases inhibition of GSK3 beta. Luprolide acetate rescues ovariectomy-dependent cognitive function, increases signaling events associated with synaptic plasticity including GSK3 beta inhibition, but does not alter AD pathology. In the human AD female brain, luteinizing hormone (LH) mRNA levels are reduced. In the 3XAD-tg model, brain LH protein levels are reduced by ovariectomy and normalized by leuprolide acetate treatment. These treatment-dependent normalization of LH positively correlates with markers of neuroplasticity and cognitive improvement.

Role of GSK3 beta and ERK in the human neutrophil response to fungal beta‐glucan (1046.5)

Complement Receptor 3 (CR3), an integrin found on neutrophils, plays an important role in the neutrophil recognition and response to fungal components in the context of extracellular matrix proteins. Co‐ligation of CR3 with fibronectin (Fn) at the I‐domain and ß‐glucan (B) at the lectin‐like domain is possible due to spatially distinct locations and creates unique cellular activity not seen with ligation of either domain separately. Dual ligation of CR3 with Fn+B induces homotypic aggregation of primed neutrophils, a response representative of the immune cells encounter with fungi within tissues. To understand the signaling pathways involved in these CR3‐mediated responses, we employed various molecular techniques to determine the key proteins. Mass spectroscopy revealed a significant increase in tyrosine phosphorylation of nineteen proteins in human neutrophil samples exposed to FN+B but not in samples exposed to FN alone. Western blotting and immunopreciptation techniques validate these findings by showing an increase in two of the proteins, p‐ERK and p‐GSK. Further work using inhibitors UO126 (ERK inhibitor) and TZDZ‐8 (GSK3Beta inhibitor), show differing effects on cluster formation. Use of UO126 caused a complete abrogation of cluster formation and a significant loss of ERK phosphorylation. However, use of TZDZ‐8 caused a partial inhibition of cluster formation with little effect on GSK3Beta phosphorylation. Blotting for protein levels, we were able to show the phosphorylation of ERK increases over time while the phophorylation of GSK decreases. Use of TZDZ‐8 decreased the phosphorylation of ERK, while inhibition with UO126 had little effect on GSK3 phosphoryaltion. This result indicates a upstream role for GSK3 Beta in the phosphorylation of ERK. In addition, monoclonal antibody blocking of CR3 led to an increase in GSK3 Beta phosphorylation indicating CR3 signaling of p‐ERK may be regulated through GSK3 Beta phosphylation.Grant Funding Source: Supported by NIH GM‐066194

Glia and immune cell signaling in bipolar disorder: insights from neuropharmacology and molecular imaging to clinical application

Bipolar disorder (BD) is a debilitating mental illness characterized by severe fluctuations in mood, sleep, energy and executive functioning. Pharmacological studies of selective serotonin reuptake inhibitors and the monoamine system have helped us to clinically understand bipolar depression. Mood stabilizers such as lithium and valproic acid, the first-line treatments for bipolar mania and depression, inhibit glycogen synthase kinase-3 beta (GSK-3β) and regulate the Wnt pathway. Recent investigations suggest that microglia, the resident immune cells of the brain, provide a physiological link between the serotonin system and the GSK-3β/Wnt pathway through neuroinflammation. We review the pharmacological, translational and brain imaging studies that support a role for microglia in regulating neurotransmitter synthesis and immune cell activation. These investigations provide a model for microglia involvement in the pathophysiology and phenotype of BD that may translate into improved therapies.

The curry spice curcumin attenuates beta-amyloid-induced toxicity through beta-catenin and PI3K signaling in rat organotypic hippocampal slice culture

Objective: Accumulating evidence indicates that curcumin potently protects against beta-amyloid (Abeta) due to its oxygen free radicals scavenging and anti-inflammatory properties. However, cellular mechanisms that may underlie the neuroprotective effect of curcumin in Abeta-induced toxicity are not fully understood yet. The present study was undertaken to investigate the mechanisms involved in neuroprotective effects of curcumin, particularly involving Wnt/beta-catenin and PI3K pathways.Methods: Organotypic hippocampal slice cultures were treated with curcumin and exposed to Abeta1–42 for 48 hours. Synaptic dysfunction, cell death, ROS formation, neuroinflammation and beta-catenin, Akt, and GSK-3beta phosphorylation were measured to determine the effects of curcumin against Abeta toxicity.Results: Curcumin significantly attenuated Abeta-induced cell death, loss of synaptophysin, and ROS generation. Furthermore, curcumin was able to decrease IL-6 release and increase IL-10 release, and prevented glial activation. The phosphorylation of beta-catenin was avoided and the levels of free beta-catenin were increased by curcumin to promote cell survival upon treatment with Abeta. Curcumin, in the presence of Abeta, activated Akt which in turn phosphorylates GSK-3beta, and resulted in the inhibition of GSK-3beta. The presence of LY294002, an inhibitor of PI3K pathway, blocked the pro-survival effect of curcumin.Discussion: These results reinforce the neuroprotective effects of curcumin on Abeta toxicity and add some evidence that its mechanism may involve beta-catenin and PI3K signaling pathway in organotypic hippocampal slice culture.

Higher proportion of inactive Gsk3β in platelets of elderly patients with bipolar disorder: an effect of treatment?

It has been postulated that mood stabilizers inhibit glycogen synthase kinase 3-beta (Gsk3β) activity, mainly through its phosphorylation on serine-9 (Ser9). However, in vivo studies addressing Gsk3β activity in patients with bipolar disorder are scarce. Here, we compare Gsk3β inactivation (as indicated by Ser9-phosphorylation) in platelets of elderly patients with bipolar disorder undergoing clinical treatment and healthy elderly adults not taking medication. Platelet samples were obtained from 37 elderly adults (bipolar disorder = 19, controls = 18). Relative changes in Gsk3β inactivation was estimated by comparing the ratios of phosphorylated Gsk3β to total Gsk3β (p-Gsk3β Ser9/Gsk3β) between the disease and control groups. Phosphorylated-Gsk3β (p < 0.001) and the p-Gsk3β Ser9/Gsk3β ratio (p = 0.006) were elevated in bipolar patients. In the bipolar disorder group, p-Gsk3β Ser9/Gsk3β was positively correlated with serum lithium levels (r = 0.478, p = 0.039). Gsk3β inactivation is higher in this group of elderly adults undergoing treatment for bipolar disorder. However, whether the treatment or the disease causes Gsk3β inactivation was confounded by the lack of an unmedicated, bipolar control group and the non-uniform treatment regimens of the bipolar disorder group. Thus, further studies should help distinguish whether Gsk3β inactivation is an effect of drug treatment or an intrinsic characteristic of bipolar disorder.

The olive constituent oleuropein triggers a postconditioning-like effect in anesthetized rabbits through GSK3beta inhibition and JAK/STAT activation

Background: Nutritional dose of oleuropein (oleu), which is the main polyphenolic constituent of olive, attenuates ischemia (isc) - reperfusion (rep) injury. Aim: We investigated the effect of pharmacological dose of oleu on infarct size, the intracellular signaling pathway changes and its potential role as postconditioning (PostC) analogue in vivo. Methods: Anesthetized male rabbits were subjected to 30-min regional myocardial isc followed by 3-hour rep. The animals were divided into 6 groups with the following additional interventions: (i) Control group: no further intervention; (ii) PostC group: 8 cycles of 30-sec isc/rep at the onset of rep; (iii) Oleu-50 group and (iv) Oleu-100 group: intravenous bolus administration of 50 and 100 mg.kg-1 oleu at the 20th min of isc, respectively; (v) Oleu-100 +L-NAME group and (vi) Oleu-100+Wortmannin group: administration of the NOS inhibitor L-NAME at a dose of 10 mg.kg-1 and of the PI3K/Akt inhibitor wortmannin at a dose of 60 μg.kg-1, respectively, one min before oleu administration. Circulating oleu plasma levels were assessed at the 1st and 20th min of rep by UHPLC-ESI-HRMS/MS. The percent infarct to area at risk ratio (%I/R) was estimated. In a second series of experiments, rabbits were subjected to the same initial interventions and then tissue samples were quickly excised from the ischemic regions of the heart at the 10th min of rep for eNOS, Akt, GSK3β and STAT3 assessment. Results: A significant %I/R reduction was observed in the Oleu-100 and PostC groups compared to the Control and Oleu-50 groups (20.0±2.5%, 26.4±0.3% vs 49.2±2.0% and 44.2±2.0 respectively, p<0.05). L-NAME and wortmannin did not abolish the cardioprotection afforded by Oleu-100 (23.8±4.42% and 24.5±2.4% respectively, p=NS vs Oleu-100 group).The phosphorylation of e-NOS, Akt, GSK3β and STAT3 was also significantly higher in the Oleu-100 and PostC groups (p<0.05 vs Control). Circulating oleu levels have been found to exhibit a 20-fold increase in the Oleu-100 group compared to the Oleu-50 group at both time points. Conclusion: Higher dose of oleu, given acutely at the index isc and prior to the onset of rep, induced a postconditioning-like effect in anesthetized rabbits, reducing the final infarct size through GSK3β inhibition and STAT3 activation, independently of eNOS and Akt. Cardioprotection from oleu is a dose-dependent effect that is possibly correlated with the increased circulating plasma levels.

Specific Induction of Akt3 in Spinal Cord Motor Neurons is Neuroprotective in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Evidence is accumulating that an imbalance between pathways for degeneration or survival in motor neurons may play a central role in mechanisms that lead to neurodegeneration in amyotrophic lateral sclerosis (ALS). We and other groups have observed that downregulation, or lack of induction, of the PI3K/Akt prosurvival pathway may be responsible for defective response of motor neurons to injury and their consequent cellular demise. Some of the neuroprotective effects mediated by growth factors may involve activation of Akt, but a proof of concept of Akt as a target for therapy is lacking. We demonstrate that specific expression of constitutively activated Akt3 in motor neurons through the use of the promoter of homeobox gene Hb9 prevents neuronal loss induced by SOD1.G93A both in vitro (in mixed neuron/astrocyte cocultures) and in vivo (in a mouse model of ALS). Inhibition of ASK1 and GSK3beta was involved in the neuroprotective effects of activated Akt3, further supporting the hypothesis that induction of Akt3 may be a key step in activation of pathways for survival in the attempt to counteract motor neuronal degeneration in ALS.

Inhibition of GSK3B Bypass Drug Resistance of p53-Null Colon Carcinomas by Enabling Necroptosis in Response to Chemotherapy

Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.