GSK-3β inhibitors suppressed neuroinflammation in rat cortex by activating autophagy in ischemic brain injury

Previous studies have shown that GSK-3β inhibitor could reduce infarct volume after ischemia brain injury. However, the underlying mechanisms of GSK-3β inhibitor involving neuroprotection remain poorly understood. In the present study, we demonstrated that GSK-3β inhibitor suppressed insult-induced neuroinflammation in rat cortex by increasing autophagy activation in ischemic injury. Male rats were subjected to pMCAO (permanent middle cerebral artery occlusion) followed by treating with SB216763, a GSK-3β inhibitor. We found that insult-induced inflammatory response was significantly decreased by intraperitoneal infusion of SB216763 in rat cortex. A higher level of autophagy was also detected after SB216763 treatment. In the cultured primary microglia, SB216763 activated autophagy and suppressed inflammatory response. Importantly, inhibition of autophagy by Beclin1-siRNA increased inflammatory response in the SB216763-treated microglia. These data suggest that GSK-3β inhibitor suppressed neuroinflammation by activating autophagy after ischemic brain injury, thus offering a new target for prevention of ischemic brain injury.     

The Involvement of Mitochondrial Biogenesis in Selenium Reduced Hyperglycemia-Aggravated Cerebral Ischemia Injury

Selenium has been shown to possess antioxidant and neuroprotective effects by modulating mitochondrial function and activating mitochondrial biogenesis. Our previous study has also suggested that selenium protected neurons against glutamate toxicity and hyperglycemia-induced damage by regulating mitochondrial fission and fusion. However, it is still not known whether the mitochondrial biogenesis is involved in selenium alleviating hyperglycemia-aggravated cerebral ischemia reperfusion (I/R) injury. The object of this study is to define whether selenium protects neurons against hyperglycemia-aggravated cerebral I/R injury by promoting mitochondrial biogenesis. In vitro oxygen deprivation plus high glucose model decreased cell viability, enhanced reactive oxygen species production, and meanwhile stimulated mitochondrial biogenesis signaling. Pretreated with selenium significantly decreased cell death and further activated the mitochondrial biogenesis signaling. In vivo 30 min of middle cerebral artery occlusion in the rats under hyperglycemic condition enhanced neurological deficits, enlarged infarct volume, exacerbated neuronal damage and oxidative stress compared with normoglycemic ischemic rats after 24 h reperfusion. Consistent to the in vitro results, selenium treatment alleviated ischemic damage in hyperglycemic ischemic animals. Furthermore, selenium reduced the structural changes of mitochondria caused by hyperglycemic ischemia and further promoted the mitochondrial biogenesis signaling. Selenium activates mitochondrial biogenesis signaling, protects mitochondrial structure integrity and ameliorates cerebral I/R injury in hyperglycemic rats.

     

25-Hydroxycholesterol induces mitochondria-dependent apoptosis via activation of glycogen synthase kinase-3β in PC12 cells

25-Hydroxycholesterol (25-OH-chol) induces apoptosis in many cell types. The present study investigated the possible involvement of mitochondria-dependent apoptotic signalling molecules in the death of PC12 cells treated with 25-OH-chol. 25-OH-chol increased the production of reactive oxygen species and opened mitochondrial permeability transition pore, resulting in release of cytochrome c and subsequent activation of caspase-9 and -3. 25-OH-chol induced the activation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3β (GSK-3β). The JNK inhibitor SP600125 attenuated the activation of caspase-9 and -3 and reduced 25-OH-chol-induced cell death. GSK inhibitors SB415286 and SB216763 significantly down-regulated JNK activity and attenuated the cytotoxicity of 25-hydroxycholesterol. However, SP600125 did not alter the activity of GSK-3β. The results indicate that 25-OH-chol induces cell death via activation of GSK-3β and subsequent up-regulation of JNK. Pharmacological intervention of GSK-3β-JNK-caspase signalling pathway may be useful for the reduction of cytotoxicity of oxysterols.     

25-hydroxycholesterol induces mitochondria-dependent apoptosis via activation of glycogen synthase kinase-3beta in PC12 cells

25-hydroxycholesterol (25-OH-chol) induces apoptosis in many cell types. The present study investigated the possible involvement of mitochondria-dependent apoptotic signalling molecules in the death of PC12 cells treated with 25-OH-chol. 25-OH-chol increased the production of reactive oxygen species and opened mitochondrial permeability transition pore, resulting in release of cytochrome c and subsequent activation of caspase-9 and -3. 25-OH-chol induced the activation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3beta (GSK-3beta). The JNK inhibitor SP600125 attenuated the activation of caspase-9 and -3 and reduced 25-OH-chol-induced cell death. GSK inhibitors SB415286 and SB216763 significantly down-regulated JNK activity and attenuated the cytotoxicity of 25-hydroxycholesterol. However, SP600125 did not alter the activity of GSK-3beta. The results indicate that 25-OH-chol induces cell death via activation of GSK-3beta and subsequent up-regulation of JNK. Pharmacological intervention of GSK-3beta-JNK-caspase signalling pathway may be useful for the reduction of cytotoxicity of oxysterols.     

Caspase-dependent Apoptosis Induced by Thapsigargin was Prevented by Glycogen Synthase Kinase-3 Inhibitors in Cultured Rat Cortical Neurons

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca²⁺-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9–10). Blockade of N-methyl-d-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3β activation in rat cortical neurons.     

Inhibition of glycogen synthase kinase-3β prevents sympathetic hyperinnervation in infarcted rats

We have demonstrated that nerve growth factor (NGF) expression in the myocardium is selectively increased during chronic stage of myocardial infarction, resulting in sympathetic hyperinnervation. Glycogen synthase kinase-3 (GSK-3) signal has been shown to play key roles in the regulation of cytoskeletal assembly during axon regeneration. We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction through attenuated NGF expression and Tau expression. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to either LiCl or SB216763, chemically unrelated inhibitors of GSK-3β, a combination of LiCl and SB216763, or vehicle for four weeks. Myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats compared with sham-operated rats, consistent with excessive sympathetic reinnervation after infarction. Immunohistochemical analysis for sympathetic nerve also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administering either LiCl, SB216763, or combination. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those treated with GSK-3 inhibitors. Addition of SB216763 did not have additional beneficial effects compared with those seen in rats treated with LiCl alone. Furthermore, lithium treatment increased Tau1 and decreased AT8 and AT180 levels. Chronic use of lithium after infarction, resulting in attenuated sympathetic reinnervation by GSK-3 inhibition, may modify the arrhythmogenic response to programmed electrical stimulation.     

Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.     

Glycogen synthase kinase 3β in the basolateral amygdala is critical for the reconsolidation of cocaine reward memory

Exposure to cocaine-associated conditioned stimuli elicits craving and increases the probability of cocaine relapse in cocaine users even after extended periods of abstinence. Recent evidence indicates that cocaine seeking can be inhibited by disrupting the reconsolidation of the cocaine cue memories and that basolateral amygdala (BLA) neuronal activity plays a role in this effect. Previous studies demonstrated that glycogen synthase kinase 3β (GSK-3β) plays a role in the reconsolidation of fear memory. Here, we used a conditioned place preference procedure to examine the role of GSK-3β in the BLA in the reconsolidation of cocaine cue memories. GSK-3β activity in the BLA, but not central amygdala (CeA), in rats that acquired cocaine (10 mg/kg)-induced conditioned place preference increased after re-exposure to a previously cocaine-paired chamber (i.e., a memory reactivation procedure). Systemic injections of the GSK-3β inhibitor lithium chloride after memory reactivation impaired the reconsolidation of cocaine cue memories and inhibited subsequent cue-induced GSK-3β activity in the BLA. Basolateral amygdala, but not central amygdala, injections of SB216763, a selective inhibitor of GSK-3β, immediately after the reactivation of cocaine cue memories also disrupted cocaine cue memory reconsolidation and prevented cue-induced increases in GSK-3β activity in the BLA. The effect of SB216763 on the reconsolidation of cocaine cue memories lasted at least 2 weeks and was not recovered by a cocaine priming injection. These results indicate that GSK-3β activity in the BLA mediates the reconsolidation of cocaine cue memories.     

Overexpression of lemur tyrosine kinase-2 protects neurons from oxygen-glucose deprivation/reoxygenation-induced injury through reinforcement of Nrf2 signaling by modulating GSK-3β phosphorylation

Lemur tyrosine kinase-2 (LMTK2), a newly identified serine/threonine kinase, is a potential regulator of cell survival and apoptosis. However, little is known about its role in regulating neuronal survival during cerebral ischemia/reperfusion injury. The present study aimed to explore the potential function of LMTK2 in regulating neuronal survival using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury. Herein, we found that LMTK2 expression was markedly decreased in neurons following OGD/R exposure. Gain-of-function experiments demonstrated that LMTK2 overexpression significantly improved the viability and reduced apoptosis of neurons with OGD/R-induced injury. Moreover, LMTK2 overexpression reduced the production of reactive oxygen species (ROS) in OGD/R-exposed neurons. Notably, our results elucidated that LMTK2 overexpression reinforced the activation of nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) antioxidant signaling associated with increased glycogen synthase kinase-3β (GSK-3β) phosphorylation. GSK-3β inhibition by its specific inhibitor significantly reversed LMTK2-inhibition-linked apoptosis and ROS production. Additionally, silencing Nrf2 partially reversed the LMTK2-overexpression-mediated neuroprotective effect in OGD/R-injured neurons. Taken together, our results demonstrated that LMTK2 overexpression alleviated OGD/R-induced neuronal apoptosis and oxidative damage by enhancing Nrf2/ARE antioxidant signaling via modulation of GSK-3β phosphorylation. Our study suggests LMTK2 is a potential target for neuroprotection during cerebral ischemia/reperfusion.     

Opposed effects of lithium on the MEK-ERK pathway in neural cells: inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms

Lithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time- and dose-dependently the phosphorylation of MEK and ERK, with 1 mM concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7-day exposure. Lithium also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serum-deprived, quiescent astrocytes, pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3-5 min, but was effective in inhibiting the long-term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic beta-catenin, consistent with the inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentration-dependent manner, again through an inositol and GSK-3 beta independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury.     

Neuronal pentraxin 1 induction in hypoxic-ischemic neuronal death is regulated via a glycogen synthase kinase-3α/β dependent mechanism

Intracellular signaling pathways that regulate the production of lethal proteins in central neurons are not fully characterized. Previously, we reported induction of a novel neuronal protein neuronal pentraxin 1 (NP1) in neonatal brain injury following hypoxia-ischemia (HI); however, how NP1 is induced in hypoxic-ischemic neuronal death remains elusive. Here, we have elucidated the intracellular signaling regulation of NP1 induction in neuronal death. Primary cortical neurons showed a hypoxic-ischemia time-dependent increase in cell death and that NP1 induction preceded the actual neuronal death. NP1 gene silencing by NP1-specific siRNA significantly reduced neuronal death. The specificity of NP1 induction in neuronal death was further confirmed by using NP1 (−/−) null primary cortical neurons. Declines in phospho-Akt (i.e. deactivation) were observed concurrent with decreased phosphorylation of its downstream substrate GSK-3α/β (at Ser21/Ser9) (i.e. activation) and increased GSK-3α and GSK-3β kinase activities, which occurred prior to NP1 induction. Expression of a dominant-negative inhibitor of Akt (Akt-kd) blocked phosphorylation of GSK-3α/β and subsequently enhanced NP1 induction. Whereas, overexpression of constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) increased GSK-α/β phosphorylation and attenuated NP1 induction. Transfection of neurons with GSK-3α siRNA completely blocked NP1 induction and cell death. Similarly, overexpression of the GSK-3β inhibitor Frat1 or the kinase mutant GSK-3βKM, but not the wild-type GSK-3βWT, blocked NP1 induction and rescued neurons from death. Our findings clearly implicate both GSK-3α- and GSK-3β-dependent mechanism of NP1 induction and point to a novel mechanism in the regulation of hypoxic-ischemic neuronal death.     

GSK-3beta inhibitors reduce protein degradation in muscles from septic rats and in dexamethasone-treated myotubes

Sepsis is associated with muscle wasting, mainly reflecting increased muscle proteolysis. Recent studies suggest that inhibition of GSK-3beta activity may counteract catabolic stimuli in skeletal muscle. We tested the hypothesis that treatment of muscles from septic rats with the GSK-3beta inhibitors LiCl and TDZD-8 would reduce sepsis-induced muscle proteolysis. Because muscle wasting during sepsis is, at least in part, mediated by glucocorticoids, we also tested the effects of GSK-3beta inhibitors on protein degradation in dexamethasone-treated cultured myotubes. Treatment of incubated extensor digitorum longus muscles with LiCl or TDZD-8 reduced basal and sepsis-induced protein breakdown rates. When cultured myotubes were treated with LiCl or one of the GSK-3beta inhibitors SB216763 or SB415286, protein degradation was reduced. Treatment of incubated muscles or cultured myotubes with LiCl, but not the other GSK-3beta inhibitors, resulted in increased phosphorylation of GSK-3beta at Ser9, consistent with inactivation of the kinase and suggesting that the other inhibitors used in the present experiments inhibit GSK-3beta by phosphorylation-independent mechanisms. The present results suggest that GSK-3beta inhibitors may be used to prevent or treat sepsis-induced, glucocorticoid-regulated muscle proteolysis.     

GSK-3 mediates the release of IL-1β, TNF-α and IL-10 from cortical glia

Neuroinflammation has been shown to contribute to neurodegenerative and psychiatric disorders such as Alzheimer's disease and major depression due to the inappropriate release of pro-inflammatory cytokines from activated microglia. The precise molecular events that mediate cytokine release from glia remain unknown but we suggest that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) may be involved. The aim of this study therefore was to investigate the effect of lipopolysaccharide (LPS) on expression and activity of the GSK-3β isoform in glia, and to assess if GSK-3 mediates the LPS-induced change in inflammatory cytokine levels in culture medium from rat glial-enriched cortical cultures. GSK-3β was expressed in microglia and astrocytes, and stimulation of these cultures with LPS induced an increase in GSK-3β expression and activity, and in pro-inflammatory cytokine levels in culture media. We show that GSK-3 inhibition using a small molecule inhibitor SB216763 or the mood stabiliser lithium chloride reduced the LPS-induced elevated levels of pro-inflammatory cytokines present in culture media from rat glial-enriched cortical cultures. These results demonstrate a role for GSK-3 as a modulator of inflammatory cytokine levels in the brain, and contribute to a mechanistic insight into neurological disorders in which neuroinflammation is a characteristic feature.     

SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD‐dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac‐p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53‐induced mitochondrial dysfunction and neuronal damage in a deacetylase activity‐dependent manner. Notably, mitochondrially targeted p53 (mito‐p53) directly reduced mitochondria DNA‐encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53‐ChIP analysis verified the presence of p53‐binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito‐p53 activity. Our results indicate that SIRT3 dysfunction leads to p53‐mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD.