The novel AKT inhibitor afuresertib suppresses human Merkel cell carcinoma MKL-1 cell growth.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine neoplasm of the skin, which has an exceedingly poor prognosis. The AKT/mammalian target of rapamycin (mTOR) signalling pathway, which plays a pivotal role in the modulation of protein synthesis and cell survival, has been shown to be extremely important for Merkel cell carcinogenesis. In the current study, we found that AKT has important regulatory functions in MCC cells and that inhibition of AKT with the novel ATP-competitive AKT inhibitor, afuresertib, has widespread effects on proliferative pathways. In particular, we found that treatment of MCC cells with afuresertib led to deactivation of mTOR and glycogen synthase kinase 3 pathway proteins while increasing activation of proapoptotic pathways through the upregulation of p16 expression and phosphomodulation of the B-cell lymphoma-2-associated death promoter. Overall, afuresertib treatment led to significant and robust inhibition of MCC cell proliferation, thus raising intriguing questions regarding the potential efficacy of AKT inhibition for the future clinical management of MCC.

Adolescent nicotine induces depressive and anxiogenic effects through ERK 1-2 and Akt-GSK-3 pathways and neuronal dysregulation in the nucleus accumbens.

Long-term tobacco dependence typically develops during adolescence and neurodevelopmental nicotine exposure is associated with affective disturbances that manifest as a variety of neuropsychiatric comorbidities in clinical and preclinical studies, including mood and anxiety-related disorders. The nucleus accumbens shell (NASh) is critically involved in regulating emotional processing, and both molecular and neuronal disturbances in this structure are associated with mood and anxiety-related pathologies. In the present study, we used a rodent model of adolescent neurodevelopmental nicotine exposure to examine the expression of several molecular biomarkers associated with mood/anxiety-related phenotypes. We report that nicotine exposure during adolescence (but not adulthood) induces profound upregulation of the ERK 1-2 and Akt-GSK-3 signalling pathways directly within the NASh, as well as downregulation of local D1R expression that persists into adulthood. These adaptations were accompanied by decreases in τ, α, ß, and γ-band oscillatory states, hyperactive medium spiny neuron activity with depressed bursting rates, and anxiety and depressive-like behavioural abnormalities. Pharmacologically targeting these molecular and neuronal adaptations revealed that selective inhibition of local ERK 1-2 and Akt-GSK-3 signalling cascades rescued nicotine-induced high-γ-band oscillatory signatures and phasic bursting rates in the NASh, suggesting that they are involved in mediating adolescent nicotine-induced depressive and anxiety-like neuropathological trajectories.

Exendin-4 Attenuates Remodeling in the Remote Myocardium of Rats After an Acute Myocardial Infarction by Activating ß-Arrestin-2, Protein Phosphatase 2A, and Glycogen Synthase Kinase-3 and Inhibiting ß-Catenin.

PURPOSE: This study tested if the protective anti-remodeling effect of GLP-1 agonist Exendin-4 after an acute myocardial infarction (MI) in rats involves inhibition of the Wnt1/ß-catenin signaling pathway. METHODS: Rats were divided into sham, sham + Exendin-4 (10 µg/day, i.p), MI, and MI + Exendin-4. MI was introduced to rats by permanent left anterior descending coronary artery (LAD) ligation. RESULTS: On day 7 post-infraction, MI rats showed LV dysfunction with higher serum levels of cardiac markers. Their remote myocardia showed increased mRNA and protein levels of collagen I/III with higher levels of reactive oxygen species (ROS) and inflammatory cytokines, as well as protein levels of Wnt1, phospho-Akt, transforming growth factor (TGF-ß1), Smad, phospho-Smad3, α-SMA, caspase-3, and Bax. They also showed higher protein levels of phospho-glycogen synthase kinase-3ß (p-GSK3ß), as well as total, phosphorylated, and nuclear ß-catenin with a concomitant decrease in the levels of cyclic adenosine monophosphate (cAMP), mRNA of manganese superoxide dismutase (MnSOD), and protein levels of Bcl-2, ß-arrestin-2, and protein phosphatase-2 (PP2A). Administration of Exendin-4 to MI rats reduced the infarct size and reversed the aforementioned signaling molecules without altering protein levels of TGF-1ß and Wnt1 or Akt activation. Interestingly, Exendin-4 increased mRNA levels of MnSOD, protein levels of ß-arrestin-2 and PP2A, and ß-catenin phosphorylation but reduced the phosphorylation of GSK3ß and Smad3, and total ß-catenin levels in the LV of control rats. CONCLUSION: Exendin-4 inhibits the remodeling in the remote myocardium of rats following acute MI by attenuating ß-catenin activation and activating ß-arrestin-2, PP2A, and GSK3ß. Graphical Abstract A graphical abstract that illustrates the mechanisms by which Exendin-4 inhibits cardiac remodeling in remote myocardium of left ventricle MI-induced rats. Mechanisms are assumed to occur in the cardiomyocytes and/or other resident cells such as fibroblast. Β-catenin activation and nuclear translocation are associated with increased synthesis of inflammatory cytokines and transforming growth factor ß-1 (TGF-ß1). GSK3ß is inhibited by phosphorylation at Ser9. Under normal conditions, ß-catenin is degraded in the cytoplasm by the active GSK3ß-dependent degradation complex (un-phosphorylated) which usually phosphorylates ß-catenin at Ser33/37/Thr41. After MI, TGF-ß1, and Wnt 1 levels are significantly increased, the overproduction of Wnt1 induces ß-catenin stabilization and nuclear translocation through increasing the phosphorylation of disheveled (DVL) protein which in turn phosphorylates and inhibits GSK3ß. TGF-ß1 stimulates the phosphorylation of Smad-3 and subsequent nuclear translocation to activate the transcription of collage 1/III and α-smooth muscle actin (α-SMA). Besides, TGF-ß1 stabilizes cytoplasmic ß-catenin levels indirectly by phosphorylation of Akt at Thr308-induced inhibition of GSK3ß by increasing phosphorylation of Ser9. Exendin-4, and possibly through G protein-coupled receptors (GPCRs), increases levels of cAMP and upregulates ß-arrestin-2 levels. Both can result in a positive inotropic effect. Besides, ß-arrestin-2 can stimulate PP2A to dephosphorylation Smad3 (inhibition) and GSK3ß (activation), thus reduces fibrosis and prevents the activation of ß-catenin and collagen deposition.

l-Theanine Prevents Long-Term Affective and Cognitive Side Effects of Adolescent Δ-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.

Chronic adolescent exposure to Δ-9-tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioral abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathologic alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. l-Theanine is an amino acid analog of l-glutamate and l-glutamine derived from various plant sources, including green tea leaves. l-Theanine has previously been shown to modulate levels of GABA, DA, and glutamate in various neural regions and to possess neuroprotective properties. Using a preclinical model of adolescent THC exposure in male rats, we report that l-theanine pretreatment before adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect that persists into adulthood. In addition, pretreatment with l-theanine blocked THC-induced downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and subcortical DAergic dysregulation. Finally, l-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long-term neuroprotective effects of l-theanine in the mesocorticolimbic system.SIGNIFICANCE STATEMENT With the increasing trend of cannabis legalization and consumption during adolescence, it is essential to expand knowledge on the potential effects of adolescent cannabis exposure on brain development and identify potential pharmacological strategies to minimize Δ-9-tetrahydrocannabinol (THC)-induced neuropathology. Previous evidence demonstrates that adolescent THC exposure induces long-lasting affective and cognitive abnormalities, mesocorticolimbic dysregulation, and schizophrenia-like molecular biomarkers that persist into adulthood. We demonstrate for the first time that l-theanine, an amino acid analog of l-glutamate and l-glutamine, is capable of preventing long-term THC side effects. l-Theanine prevented the development of THC-induced behavioral aberrations, blocked cortical downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways, and normalized dysregulation of both PFC and VTA DAergic activity, demonstrating powerful and functional neuroprotective effects against THC-induced developmental neuropathology.

Maternal nicotine exposure has severe cross-generational effects on offspring behavior.

Our previous studies showed that paternal nicotine exposure can lead to hyperactivity in the offspring. Nevertheless, the cross-generational effects of maternal and biparental nicotine exposure remain unclear. In this study, female and male mice were exposed respectively by nicotine before pregnancy. The maternal pre-pregnancy nicotine exposure led to depression-like behaviors in the F1 offspring. However, after biparental pre-pregnancy nicotine exposure, seventy percentage of the offspring exhibited a depressive phenotype while 20% were hyperactive, and the remaining exhibited no obvious abnormal behavior. The cross-generational effects appeared to be mediated via disruption of the balance between GSK3 and p-GKS3 by nicotine. These results suggested that pre-pregnancy nicotine exposure can induce alterations in the behavior of the offspring, and the cross-generational effects of maternal nicotine exposure were particularly serious.

The role of histone deacetylase inhibitors in regulation of Akt/GSK-3ß signaling pathway in mice following transient focal cerebral ischemia.

PURPOSE: To investigate whether the neuroprotective effect of TSA on cerebral ischemia reperfusion injury is mediated by the activation of Akt/GSK-3ß signaling pathway. METHODS: Mice were randomly divided into four groups (n=15): sham group (S); ischemia reperfusion group (IR); ischemia reperfusion and pretreated with TSA group (IR+T); ischemia reperfusion and pretreated with TSA and LY294002 group (IR+T+L). The model of cerebral ischemia reperfusion was established by 1h of MCAO following 24h of reperfusion. TSA (5mg/kg) was intraperitoneally given for 3 days before MCAO, Akt inhibitor, LY294002 (15 nmol/kg) was injected by tail vein 30 min before the MCAO. RESULTS: TSA significantly increased the expression of p-Akt, p-GSK-3ß proteins and the levels of SOD, Bcl-2, reduced the infarct volume and the levels of MDA, ROS, TNF-α, IL-1ß, Bax, Caspase-3, TUNEL and attenuated neurological deficit in mice with transient MCAO, LY294002 weakened such effect of TSA dramatically. CONCLUSIONS: TSA could significantly decrease the neurological deficit and reduce the cerebral infarct volume, oxidative stress, inflammation, as well as apoptosis during cerebral ischemia reperfusion injury, which was achieved by activation of the Akt/GSK-3ß signaling pathway.

The Role of Histone Deacetylase Inhibitors in Regulation of Akt/GSK-3ß Signaling Pathway in Mice Following Transient Focal Cerebral Ischemia

Abstract Purpose: To investigate whether the neuroprotective effect of TSA on cerebral ischemia reperfusion injury is mediated by the activation of Akt/GSK-3β signaling pathway. Methods: Mice were randomly divided into four groups (n=15): sham group (S); ischemia reperfusion group (IR); ischemia reperfusion and pretreated with TSA group (IR+T); ischemia reperfusion and pretreated with TSA and LY294002 group (IR+T+L). The model of cerebral ischemia reperfusion was established by 1h of MCAO following 24h of reperfusion. TSA (5mg/kg) was intraperitoneally given for 3 days before MCAO, Akt inhibitor, LY294002 (15 nmol/kg) was injected by tail vein 30 min before the MCAO. Results: TSA significantly increased the expression of p-Akt, p-GSK-3β proteins and the levels of SOD, Bcl-2, reduced the infarct volume and the levels of MDA, ROS, TNF-α, IL-1β, Bax, Caspase-3, TUNEL and attenuated neurological deficit in mice with transient MCAO, LY294002 weakened such effect of TSA dramatically. Conclusions: TSA could significantly decrease the neurological deficit and reduce the cerebral infarct volume, oxidative stress, inflammation, as well as apoptosis during cerebral ischemia reperfusion injury, which was achieved by activation of the Akt/GSK-3β signaling pathway.

Thiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels.

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200mg/kg/day in drinking water for 20days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3ß (GSK-3ß) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders.

Early effects of Aß1-42 oligomers injection in mice: Involvement of PI3K/Akt/GSK3 and MAPK/ERK1/2 pathways.

Neuronal and synaptic loss are the best pathological correlates for memory decline in Alzheimer's disease (AD). Soluble beta-amyloid oligomers (AßO) are considered to putatively play a crucial role in the early synapse loss and cognitive impairment observed in AD. Evidence suggests that oxidative stress and apoptosis are involved in the mechanism of Aß-induced neurotoxicity and AD pathogenesis. This study aimed to explore the molecular mechanisms that contribute to the early memory deficits induced by intracerebroventricular injection of AßO in mice. Ten days after a single AßO injection memory impairments were observed, as measured by Morris water maze and novel object recognition tests. Cognitive decline was associated with increased oxidative stress, caspase-9 activation, and decreased hippocampal synaptophysin immunoreactivity. Furthermore, GSH levels were significantly higher in AßO-injected mice than in sham mice, showing that a protective mechanism might develop due to oxidative stress. Additionally, AßO-induced toxicity was aligned with an increment of the activation of Akt and ERK1/2, and reduced activity of GSK3. These findings suggest that AßO injection triggers a cascade of events that mimic the key neuropathological hallmarks of AD. Aß acute injection helps to better understand how this peptide impairs specific signaling pathways leading to synaptic and memory dysfunctions. Thus, this model is a valid tool for investigating AD and may suggest a new way to develop neuroprotective therapies at such early stages of the disease.

Nanoparticle Delivered Human Biliverdin Reductase-Based Peptide Increases Glucose Uptake by Activating IRK/Akt/GSK3 Axis: The Peptide Is Effective in the Cell and Wild-Type and Diabetic Ob/Ob Mice.

Insulin's stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2) peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h). The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK's downstream effector, Akt-GSK3-(α, ß) axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptides in vitro and in animal models for treatment of diabetes.

Neuregulin improves response to glucose tolerance test in control and diabetic rats.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

The Phosphorylation State of GSK3ß Serine 9 Correlated to the Development of Valproic Acid-Associated Fetal Cardiac Teratogenicity, Fetal VPA Syndrome, Rescued by Folic Acid Administration.

The effects of the phosphorylation state of the glycogen synthase kinase 3ß involved in the cardiac myocytes (jelly-like cells) epithelial-mesenchymal transition-associated migration during heart-valve formation were examined through the valproic acid-induced cardiac teratogenicity of transgenic line A34 of Tg in a the Brachydanio rerio embryo model. Valproic acid is an effective anti-epileptic drug; however, when taken by pregnant women to treat epilepsy, it can produce cardiac developmental defects in fetuses. In this study, the role of glycogen synthase kinase 3ß in valproic acid-induced cardiac teratogenicity was investigated. Transgenic line A34 of zebrafish embryos was used at 3 days postfertilization. The results show that 78% (18/23) of the embryos treated with 0.10 mM valproic acid (group A) had incomplete chamber formation with normal looping and 22 % (5/23) had abnormal looping. Bradycardia was also found in comparison with control embryos (P < 0.001). For the embryos treated with 0.25 mM valproic acid (group B), 92% (22/24) demonstrated chamber formation failure and looping abnormality. Pericardial effusion, noncontracting ventricles, and enlarged, slowly beating atriums were observed at 6 days postfertilization. Valproic acid inhibited phosphorylation of serine 9 in glycogen synthase kinase 3ß in a dose-dependent manner. According to immunochemical staining results, valproic acid was shown to inhibit the mass migration and proliferation of cardiomyocytes in the development of the heart-valve region through inhibition of the GSK3ß Ser 9 phosphorylation. Folic acid rescued the GSK3ß Ser 9 phosphorylation and reversed the valproic acid-induced cardiac morphological, functional, and biochemical defects.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

Insulin Signaling in Bupivacaine-induced Cardiac Toxicity: Sensitization during Recovery and Potentiation by Lipid Emulsion.

BACKGROUND: The impact of local anesthetics on the regulation of glucose homeostasis by protein kinase B (Akt) and 5'-adenosine monophosphate-activated protein kinase (AMPK) is unclear but important because of the implications for both local anesthetic toxicity and its reversal by IV lipid emulsion (ILE). METHODS: Sprague-Dawley rats received 10 mg/kg bupivacaine over 20 s followed by nothing or 10 ml/kg ILE (or ILE without bupivacaine). At key time points, heart and kidney were excised. Glycogen content and phosphorylation levels of Akt, p70 s6 kinase, s6, insulin receptor substrate-1, glycogen synthase kinase-3ß, AMPK, acetyl-CoA carboxylase, and tuberous sclerosis 2 were quantified. Three animals received Wortmannin to irreversibly inhibit phosphoinositide-3-kinase (Pi3k) signaling. Isolated heart studies were conducted with bupivacaine and LY294002-a reversible Pi3K inhibitor. RESULTS: Bupivacaine cardiotoxicity rapidly dephosphorylated Akt at S473 to 63 ± 5% of baseline and phosphorylated AMPK to 151 ± 19%. AMPK activation inhibited targets downstream of mammalian target of rapamycin complex 1 via tuberous sclerosis 2. Feedback dephosphorylation of IRS1 to 31 ± 8% of baseline sensitized Akt signaling in hearts resulting in hyperphosphorylation of Akt at T308 and glycogen synthase kinase-3ß to 390 ± 64% and 293 ± 50% of baseline, respectively. Glycogen accumulated to 142 ± 7% of baseline. Irreversible inhibition of Pi3k upstream of Akt exacerbated bupivacaine cardiotoxicity, whereas pretreating with a reversible inhibitor delayed the onset of toxicity. ILE rapidly phosphorylated Akt at S473 and T308 to 150 ± 23% and 167 ± 10% of baseline, respectively, but did not interfere with AMPK or targets of mammalian target of rapamycin complex 1. CONCLUSION: Glucose handling by Akt and AMPK is integral to recovery from bupivacaine cardiotoxicity and modulation of these pathways by ILE contributes to lipid resuscitation.

Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3ß-C/EBPß pathway.

Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPß. Specifically, IL-17 causes GSK-3ß-dependent phosphorylation of C/EBPß, which is associated with diminished C/EBPß binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3ß level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.

PBA regulates neurogenesis and cognition dysfunction after repeated electroconvulsive shock in a rat model.

Electroconvulsive therapy (ECT) was widely used to treat the refractory depression. But ECT led to the cognitive deficits plaguing the depression patients. The underlying mechanisms of the cognitive deficits remain elusive. Repeated electroconvulsive shock (rECS) was used to simulate ECT and explore the mechanisms of ECT during the animal studies. Previous studies showed rECS could lead to neurogenesis and cognitive impairment. But it was well known that neurogenesis could improve the cognition. So these suggested that the mechanism of the cognitive deficit after rECS was very complex. In present study, we explored the probable mechanisms of the cognitive deficit after rECS from neurogenesis aspect. We found the cognitive deficit was reversible and neurogenesis could bring a long-term beneficial effect on cognition. Astrogliosis and NR1 down-regulation probably participated in the reversible cognitive deficits after rECS. Phenylbutyric acid (PBA), generally as an agent to investigate the roles of histone acetylation, could prevent the reversible cognitive dysfunction, but PBA could diminish the long-term effect of enhanced cognition by rECS. These suggested that ECT could possibly bring the long-term beneficial cognitive effect by regulating neurogenesis.

Tideglusib protects neural stem cells against NMDA receptor overactivation.

BACKGROUND: N-methyl-d-aspartate (NMDA) receptors are major pharmacological targets to prevent or reduce the progression of neurodegenerative diseases. Successful therapy with NMDA receptor antagonists in humans has been limited by the severe side effects of complete receptor blockade. The aim of the present study was to investigate the possible protective effects of tideglusib against NMDA receptor overactivation in neural stem cells. METHODS: We measured the alteration in membrane integrity, free radical generation, intracellular Ca(2+) accumulation, mitochondrial membrane potential (MMP)/mitochondrial morphology and glycogen synthase kinase-3 (α/ß isoforms and phospho-GSK-3α/ß) protein expression levels following treatments. RESULTS: NMDA treatment, with or without d-serine, significantly increased LDH leakage and triggered cell death in neural stem cells. Reactive oxygen species (ROS) formation and intracellular Ca(2+) levels were increased following NMDA receptor overactivation. The significant reduction in MMP was found in NMDA/d-serine-treated cells. Tideglusib significantly decreased ROS production and membrane degradation, but did not change intracellular Ca(2+) levels following NMDA receptor activation. Both in the presence or in the absence of NMDA/d-serine, tideglusib increased MMP and the levels of phospho-GSK-3ß in NSCs. Moreover, GW9662 (a peroxisome-proliferator-activated receptor gamma (PPARγ) antagonist) treatment significantly inhibited the protective effect of tideglusib in NMDA/d-serine-treated cells. CONCLUSION: Our study provides the evidence that GSK-3ß and PPARγ may be directly involved in pathways leading to NMDA receptor-induced cell death and that the inhibitors including tideglusib may exert neuroprotective effect against these receptor overactivation.

Effects of antipsychotic drugs on the expression of synapse-associated proteins in the frontal cortex of rats subjected to immobilization stress.

The present study examined the effects of antipsychotic drugs on the expression of synapse-associated proteins in the frontal cortex of rats with and without immobilization stress. Rats were subjected to immobilization stress 6h/day for 3 weeks. The effects of atypical antipsychotic drugs, olanzapine and aripiprazole, on expression of serine(9)-phosphorylated GSK-3ß, ß-catenin, BDNF, PSD-95, and synaptophysin were determined by Western blotting. A typical antipsychotic drug, haloperidol, was used for comparison. Immobilization stress significantly decreased the expression of these proteins in the frontal cortex. Chronic administration of olanzapine and aripiprazole significantly attenuated the immobilization stress-induced decrease in the levels of these proteins, whereas haloperidol had no such effect. Additionally, olanzapine and aripiprazole significantly increased levels of phosphorylated GSK-3ß under normal conditions without stress, and aripiprazole also increased BDNF levels under this condition. These results indicate that olanzapine and aripiprazole, and, haloperidol, differentially regulate the levels of synapse-associated proteins in the rat frontal cortex. These findings may contribute to explain the neurobiological basis of how olanzapine and aripiprazole up-regulated synapse-associated proteins.

Glycogen synthase kinase-3 beta (GSK-3ß) signaling: Implications for Parkinson's disease.

Glycogen synthase kinase 3 (GSK-3) dysregulation plays an important role in the pathogenesis of numerous disorders, affecting the central nervous system (CNS) encompassing both neuroinflammation and neurodegenerative diseases. Several lines of evidence have illustrated a key role of the GSK-3 and its cellular and molecular signaling cascades in the control of neuroinflammation. Glycogen synthase kinase 3 beta (GSK-3ß), one of the GSK-3 isomers, plays a major role in neuronal apoptosis and its inhibition decreases expression of alpha-Synuclein (α-Synuclein), which make this kinase an attractive therapeutic target for neurodegenerative disorders. Parkinson's disease (PD) is a chronic neurodegenerative movement disorder characterized by the progressive and massive loss of dopaminergic neurons by neuronal apoptosis in the substantia nigra pars compacta and depletion of dopamine in the striatum, which lead to pathological and clinical abnormalities. Thus, understanding the role of GSK-3ß in PD will enhance our knowledge of the basic mechanisms underlying the pathogenesis of this disorder and facilitate the identification of new therapeutic avenues. In recent years, GSK-3ß has been shown to play essential roles in modulating a variety of cellular functions, which have prompted efforts to develop GSK-3ß inhibitors as therapeutics. In this review, we summarize GSK-3 signaling pathways and its association with neuroinflammation. Moreover, we highlight the interaction between GSK-3ß and several cellular processes involved in the pathogenesis of PD, including the accumulation of α-Synuclein aggregates, oxidative stress and mitochondrial dysfunction. Finally, we discuss about GSK-3ß inhibitors as a potential therapeutic strategy in PD.

9-Hydroxycanthin-6-one, a ß-Carboline Alkaloid from Eurycoma longifolia, Is the First Wnt Signal Inhibitor through Activation of Glycogen Synthase Kinase 3ß without Depending on Casein Kinase 1α.

Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/ß-catenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/ß-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased ß-catenin and increased phosphorylated ß-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of ß-catenin by 1 was suppressed by GSK3ß-siRNA, while compound 1 decreased ß-catenin even in the presence of CK1α-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3ß independent of CK1α.