Atorvastatin inhibits GSK-3β phosphorylation by cardiac hypertrophic stimuli

In this study we examined the effect of the statin atorvastatin on the Akt/GSK-3β pathway. Our findings indicate that atorvastatin treatment for 15 days inhibited pressure overload-induced cardiac hypertrophy and prevented nuclear translocation of GATA4 and c-Jun and AP-1 DNA-binding activity. In addition, atorvastatin treatment prevented the increase in the phosphorylation of Akt and GSK-3β caused by cardiac hypertrophy, and this effect correlated with an increase in protein levels of phosphatase and tensin homolog on chromosome 10 (PTEN), which negatively regulates the phosphoinositide-3 kinase/Akt pathway. To test whether the inhibitory effect of atorvastatin on Akt and GSK-3β phosphorylation was direct we performed in vitro studies using embryonic rat heart-derived H9c2 cells, human AC16 cardiomyoblasts and neonatal rat cardiomyocytes. Preincubation of cells with atorvastatin prevented Akt/GSK-3β phosphorylation by different hypertrophic stimuli without affecting PTEN protein levels. However, atorvastatin prevented endogenous reactive oxygen species (ROS) generation and PTEN oxidation, a process that correlates with its inactivation, suggesting that atorvastatin prevents ROS-induced PTEN inactivation in acute treatments. These findings point to a new potential anti-hypertrophic effect of statins, which can prevent activation of the Akt/GSK-3β hypertrophic pathway by modulating PTEN activation by different mechanisms in chronic and acute treatments.     

Adiponectin Attenuates Streptozotocin-Induced Tau Hyperphosphorylation and Cognitive Deficits by Rescuing PI3K/Akt/GSK-3β Pathway

Clinical studies have demonstrated that decreased adiponectin is associated with the development of Type 2 diabetes mellitus (T2DM) and Alzheimer’s disease (AD). We focused on determining the neuroprotective effect offered by adiponectin against streptozotocin-induced brain damage in ICV-STZ rat model. We found that adiponectin supplements significantly restored the cognitive deficits in ICV-STZ rat model including shorter escape latency, more crossing times and increased time spent in the target quadrant. Adiponectin supplements also increased number of dendritic branches and mushroom percentage. In addition, adiponectin supplements attenuated tau hyperphosphorylation at multiple AD-related sites through activation of protein Ser9-phosphorylated glycogen synthase kinase-3β (Ser9-GSK-3β) with increased the Akt and PI3K activity. Our data suggest that adiponectin supplements have neuroprotective effects on the ICV-STZ rat model, which may be mediated by the activation of the PI3K/Akt/GSK-3β signaling pathway.     

Extracellular enzymatically synthesized glycogen promotes osteogenesis by activating osteoblast differentiation via Akt/GSK-3β signaling pathway

Glycogen is the stored form of glucose and plays a major role in energy metabolism. Recently, it has become clear that enzymatically synthesized glycogen (ESG) has biological functions, such as the macrophage-stimulating activity. This study aimed to evaluate the effect of ESG on osteogenesis. MC3T3-E1 cells were cultured with ESG, and their cell proliferative activity and osteoblast differentiation were measured. An in vivo study was conducted in which ESG pellets with BMP-2 were grafted into mouse calvarial defects and histomorphometrically analyzed for the new bone formation. To confirm the effect of ESG on bone growth in vivo, ESG was orally administered to pregnant mice and the femurs of their pups were examined. We observed that ESG stimulated cell proliferation and enhanced messenger RNA expression of osteocalcin and osteopontin in MC3T3-E1 cells. ESG was taken up by the cells associated with GLUT-1 and activated the Akt/GSK-3β pathway. In vivo, the new bone formation in the calvarial defect was significantly accelerated by ESG and the maternal administration of ESG promoted fetal bone growth. In conclusion, ESG stimulates cell proliferation and differentiation of preosteoblasts via the activation of Akt/GSK-3β signaling and promotes new bone formation in vivo, suggesting that ESG could be a useful stimulant for osteogenesis.     

miR-219-5p inhibits tau phosphorylation by targeting TTBK1 and GSK-3β in Alzheimer's disease

As the most common neurodegenerative disease, Alzheimer's disease (AD) is characterized by memory, perception, and behavioral damage, which may ultimately lead to emotional fluctuation and even lethal delirium. Increasing studies indicate that microRNAs (miRNAs) are associated with pathological features of AD. However, the role of miR-219-5p in AD progression is still unclear. In this study, the functions of miR-219-5p were analyzed in vitro and in vivo. miR-219-5p was notably overexpressed in brain tissues of patients with AD. The overexpression of miR-219-5p activated the phosphorylation of Tau-Ser198, Tau-Ser199, Tau-Ser201, and Tau-Ser422. We further showed that miR-219-5p could mediate a decrease in the protein levels of tau-tubulin kinase 1 (TTBK1) and glycogen synthase kinase 3β (GSK-3β) by directly binding to their 3′-untranslated region, thereby promoting the phosphorylation of tau in SH-SY5Y Cells. Rescue experiments further revealed that the phosphorylation of tau-mediated by miR-219-5p was dependent on the inhibition of TTBK1 and GSK-3β. Moreover, suppressing the expression of both TTBK1 and GSK-3β using miR-219-5p remarkably rescued AD-like symptoms in amyloid precursor protein/presenilin 1 mice. Our findings indicate that the upregulation of TTBK1 and GSK-3β mediated by the loss of miR-219-5p is a possible mechanism that contributes to tau phosphorylation and AD progression.     

PKCβ Positively Regulates RANKL-Induced Osteoclastogenesis by Inactivating GSK-3β

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-κB ligand (RANKL) signaling has remained elusive. We now demonstrate that PKCβ acts as a positive regulator which inactivates glycogen synthase kinase-3β (GSK-3β) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, PKCβ expression is increased by RANKL. Pharmacological inhibition of PKCβ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-3β was decreased by PKCβ inhibition. Likewise, down-regulation of PKCβ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-3β phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the PKCβ pathway, leading to GSK-3β inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for PKCβ’s therapeutic targeting to treat inflammation-related bone diseases.     

Relationship of Adult Neurogenesis with Tau Phosphorylation and GSK-3β Activity in Subventricular Zone

Altered neurogenesis has been reported in Alzheimer disease (AD), the most common neurodegenerative disorder characterized with hyperphosphorylated tau and accumulation of β-amyloid (Aβ). Recent studies suggest that tau phosphorylation is essential for hippocampal neurogenesis, however, it is not known whether tau phosphorylation also play a role in neurogenesis of subventricular zone (SVZ), another main progenitor niche in the brain. Here, we examined the expression of phosphorylated tau (p-tau) in SVZ and analyzed the role of p-tau in adult SVZ neurogenesis. We found that the expression of p-tau increased during postnatal development and remains at a high level until adulthood, and the p-tau was colocalized with some SVZ neural precursors. However, up-regulating glycogen synthase kinase-3 (GSK-3), a crucial tau kinase, had no effect on SVZ neurogenesis in adult rat brain. The SVZ neurogenesis was also unaffected in tau knockout and human tau transgenic mice. These results suggest that tau phosphorylation and GSK-3 activation may not be essential for adult SVZ neurogenesis.     

miR-1290 promotes IL-8-mediated vascular endothelial cell adhesion by targeting GSK-3β

Background

MicroRNA-1290 (miR-1290) has been reported to be involved in many diseases and play a key role during the development process. However, the role of miR-1290 in atherosclerosis (AS) is still unclear.

Methods and results

The current study showed that the expressions of miR-1290 were high in serum of patients with hyperlipidemia. The functional role of miR-1290 were then investigated in human umbilical vein endothelial cells (HUVECs). Here, we found that miR-1290 expressions were notably enhanced in HUVECs mediated by IL-8. miR-1290 inhibitor repressed monocytic THP-1 cells adhesion to HUVECs by regulating ICAM-1 and VCAM-1, inhibited proliferation through regulating cyclinD1 and PCNA, and inhibited inflammatory response by regulating IL-1β. Mechanistically, we verified that miR-1290 mimic was able to directly target the 3′-UTR of GSK-3β mRNA using luciferase reporter assay. Knockdown of GSK-3β (si-GSK-3β) promoted HUVECs adhesion and the expression of IL-1β, and partially restore the depression effect of miR-1290 inhibitor on HUVECs adhesion and inflammation. In contrast, si-GSK-3β inhibited the proliferation of HUVECs and the expression of cyclinD1 and PCNA.

Conclusions

In summary, our study revealed that miR-1290 promotes IL-8-mediated the adhesion of HUVECs by targeting GSK-3β. However, GSK-3β is not the target protein for miR-1290 to regulate the proliferation of HUVECs. Our findings may provide potential target in atherosclerosis treatment.

     

Hyperphosphorylation of Tau Induced by Naturally Secreted Amyloid-β at Nanomolar Concentrations Is Modulated by Insulin-dependent Akt-GSK3β Signaling Pathway

Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.     

Proteasome Inhibition-Induced Downregulation of Akt/GSK-3β Pathway Contributes to Abnormality of Tau in Hippocampal Slice

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased the binding capacity of Akt-Hsp90 while increased the Akt-PP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology.     

The Alzheimer's therapeutic PBT2 promotes amyloid-β degradation and GSK3 phosphorylation via a metal chaperone activity

Impaired metal ion homeostasis causes synaptic dysfunction and treatments for Alzheimer's disease (AD) that target metal ions have therefore been developed. The leading compound in this class of therapeutic, PBT2, improved cognition in a clinical trial with AD patients. The aim of the present study was to examine the cellular mechanism of action for PBT2. We show PBT2 induces inhibitory phosphorylation of the α- and β-isoforms of glycogen synthase kinase 3 and that this activity is dependent on PBT2 translocating extracellular Zn and Cu into cells. This activity is supported when Aβ:Zn aggregates are the source of extracellular Zn and adding PBT2 to Aβ:Zn preparations promotes Aβ degradation by matrix metalloprotease 2. PBT2-induced glycogen synthase kinase 3 phosphorylation appears to involve inhibition of the phosphatase calcineurin. Consistent with this, PBT2 increased phosphorylation of other calcineurin substrates, including cAMP response element binding protein and Ca2?/calmodulin-dependent protein kinase. These data demonstrate PBT2 can decrease Aβ levels by sequestering the Zn that promotes extracellular formation of protease resistant Aβ:Zn aggregates, and that subsequent intracellular translocation of the Zn by PBT2 induces cellular responses with synapto-trophic potential. Intracellular translocation of Zn and Cu via the metal chaperone activity of PBT2 may be an important mechanism by which PBT2 improves cognitive function in people with AD.     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.     

GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein

Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson''s disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3β because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3β. GSK-3β-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3β to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3β-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3β-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3β directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3β. Together, these data establish a novel upstream role for GSK-3β as one of several kinases associated with PTMs of key proteins known to be causal in PD.After Alzheimer''s disease (AD), Parkinson''s disease (PD) is the second most prevalent neurodegenerative disease, characterized by selective loss of TH+ DA-neurons of substantia nigra (SN) with diminished production of dopamine (DA).¹ Genome-wide studies have identified SNCA and MAPT, genes encoding α-synuclein (α-Syn) and Tau, respectively, as having strong association to the genesis of PD.^{2, 3, 4} Although the precise etiology of PD remains a mystery, SNCA amplifications and mutations directly link α-Syn dysfunction to disease causation,^{5, 6} firmly establishing a role for α-Syn in sporadic and familial PD, respectively. α-Syn can be phosphorylated at several sites,⁷ and the predominance of α-Syn phosphorylated at serine 129 (S129) in Lewy bodies⁸ suggests its phosphorylation status at S129 has an important pathological role. Various PD models have shown that phosphorylation at S219 enhanced α-syn toxicity resulting in accelerated motor abnormalities and loss of DA-neurons.^{9, 10}Fewer studies have examined the role of Tau (or p-Tau) in PD, but interest in the field has grown since completion of several genome-wide association studies. p-Tau has been found to colocalize with α-Syn in tissue from sporadic PD and dementia with Lewy bodies.¹¹ We^{12, 13} and others^14,15 have also identified p-Tau in different brain regions of PD, dementia with Lewy bodies, and AD. High levels of p-Tau have also been observed in vivo in several toxin^{16, 17, 18} and transgenic α-Syn models of PD,^19,20 suggesting that p-Tau may be an important common factor in the neurodegeneration of not only tauopathies but also of synucleinopathies, such as PD.^{21, 22, 23, 24} Most studies to date have focused on the formation and accumulation of Tau and p-Tau in idiopathic PD. Yet several studies have provided evidence that leucine-rich repeat kinase-2 (LRRK2), a kinase, that when mutated is involved in familial forms of PD, can directly interact with, and activate GSK-3β, resulting in increased p-TAU formation.^25,26Among the kinases known to hyperphosphorylate Tau, glycogen synthase kinase-3β (GSK-3β) may be the most important given its ability to phosphorylate Tau at the majority of its serine/threonine sites that cause associated toxicities in AD.^27,28 The importance of GSK-3β is illustrated in that it is embryonically lethal when knocked out in mice. Regulation of GSK-3β is tightly controlled through a series of direct and indirect measures. Direct regulation occurs through autophosphorylation at Tyr216,^29,30 resulting in a kinase-active form, p-GSK-3β-Y216, whereas phosphorylation at Ser9 results in a kinase-inactive state.³¹ The activity of GSK-3β can also be controlled indirectly through binding to inhibitory complexes with other cytoplasmic proteins,^32,33 or through Wnt-mediated sequestration into multivesicular bodies³⁴ resulting in the physical separation of GSK-3β from its cytoplasmic targets. Control of GSK-3β in the normal state is therefore tightly regulated, with its dysregulation and ensuing aberrant phosphorylation of targets being a common occurrence in many diverse diseases. Several studies have shown that GSK-3β is an important mediator in the injury and repair processes of neurons during cross-talk between DA-neurons and reactive astrocytes.^35,36 These studies showed that astrocyte-derived Wnt1 was capable of blocking GSK-3β activation, allowing the nuclear accumulation of β-catenin and subsequent gene expression of β-catenin-dependent targets essential for neuron survival and repair during chemical or metabolic insults. The importance of regulating the active/inactive states of GSK-3β in regard to neuronal stability is further supported through the analysis of conditional (Tet-inducible) transgenic mice expressing a dominant-negative GSK-3β-K85R mutant or expressing the GSK-3β-S9A mutant.^37,38 In these studies, post-natal Tet-regulated expression of either GSK-3β-K85R or GSK-3β-S9A led to neurodegeneration in the cortex, striatum, and hippocampus. What separates our TG PD model from the tet-inducible GSK-3β models is the spatial patterns of transgene expression, which is influenced by the choice of promoters. The Tet-inducible GSK-3β models are expressed using a CAMKII promoter with our human(h) GSK-3β-S9A transgene being expressed under the Thy-1 promoter. CAMKII-driven expression is limited to neurons originating from the forebrain with Thy-1 promoter-driven expression restricted to neurons in all or most brain regions.^39,40 Although promoter choice effecting tissue expression ultimately decides which regions show degeneration, the important message is that both inactive and hyperactive states of GSK-3β reduce neuronal viability.In our past studies in various in vitro and in vivo models of PD and in postmortem PD tissues, we have consistently observed a positive correlation between increased α-Syn and p-Tau levels with increased GSK-3β-Y216 (the kinase-active form of GSK-3β).^{12, 13, 16, 19, 20} In in vitro studies of MPTP-treated SH-SY5Y cells, blockade of GSK-3β with lithium, or with the highly selective non-ATP competitive inhibitor, TDZD-8, prevented the induction of p-GSK-3β-Y216, abolished p-Tau formation, and reversed the accumulation and aggregation of both p-Tau and α-Syn, averting cell death.¹⁶ Other studies using Rotenone or MPTP/MPP+ in chemical PD models, have shown similar results of decreased neuronal viability during treatments accompanied by dose- and time-dependent increases in GSK-3β activation, with decreased cytotoxicity detected when GSK-3β was inhibited or knocked-down through the use of GSK-3β-specific small molecule inhibitors or through RNAi.^41,42 This suggested to us that p-GSK-3β-Y216 may have a contributory role in the pathogenesis of PD. Using a mouse model overexpressing hGSK-3β-S9A under the Thy-1 promoter together with in vitro kinase assays allowed us to discern the role GSK-3β has in the development of PD-like pathology.⁴³ Analysis of our hGSK-3β-S9A mouse model showed here for the first time that upon aging, these mice develop the cardinal features of parkinsonism, manifested as impaired motor behavior, with associated loss of TH+ neurons, reduced DA production, and shrinkage of SN. Invitro kinase assays confirmed that hGSK-3β was capable of phosphorylating α-Syn on Serine 129 together with the known ability to phosphorylate Tau. Remarkably, both α-Syn and Tau influenced the rate and magnitude of phosphorylation of the other by GSK-3β indicating that an intimate physical relationship exist between the trio of PD related proteins. Together, these data shown indicate the importance of GSK-3β activation, in the behavioral and physiological development of PD like pathology in a new mouse model.     

Discovery of potent and bioavailable GSK-3β inhibitors

Here we report on the discovery of a series of maleimides which have high potency and good selectivity for GSK-3β. The incorporation of polar groups afforded compounds with good bioavailability. The most potent compound 34 has an IC₅₀ of 0.6 nM for GSK-3β, over 100-fold selectivity against a panel of other kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3β protein with 34 bound revealed the binding mode of the template and provided insights for future optimization opportunities.     

Diverse regulation of AKT and GSK-3β by O-GlcNAcylation in various types of cells

Protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) are major components of insulin-AKT signaling that plays crucial roles in various types of tissue. Recent studies found that these two kinases are modified posttranslationally by O-GlcNAcylation. Here, we demonstrate that O-GlcNAcylation regulated phosphorylation/activation of AKT and GSK-3β in different manners in kidney HEK-293FT cells, but did not affect these two kinases in hepatic HepG2 cells. In neuronal cells, O-GlcNAcylation regulated phosphorylation of AKT negatively, but had no effect on GSK-3β. These results suggest protein-specific and cell type-specific regulation of AKT and GSK-3β by O-GlcNAcylation. Therefore, studies on the roles of AKT and GSK-3β O-GlcNAcylation should be done in a tissue- and cell type-specific manner.     

Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia

Amyloid β-peptide (Aβ), the pathogenic agent of Alzheimer disease, is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular Aβ is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of Aβ in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular Aβ, i.e. exosomes drive conformational changes in Aβ to form nontoxic amyloid fibrils and promote uptake of Aβ by microglia. The Aβ internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed Aβ incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced Aβ uptake into microglial cells and significantly decreased extracellular levels of Aβ. Our findings indicate a novel mechanism responsible for clearance of Aβ through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD.     

Effect of aluminum combined with ApoEε4 on Tau phosphorylation and Aβ deposition

BackgroundAluminum is an environmental neurotoxin widely exposed to animals and humans. Studies have shown that Alzheimer's disease (AD) is characterized by abnormally phosphorylated tau and Aβ deposition, aluminum exposure can lead to abnormal phosphorylated tau and Aβ deposition. Numerous epidemiological data and studies have confirmed that ApoEε4 is a risk factor for AD. However, whether there is an interaction effect between aluminum and ApoEε4 has yet to be verified.MethodsSH-SY5Y cells were exposed with AlCl₃ and transfected with ApoEε4 respectively. The experimental groups included the blank control group, the low dose group (200 μM AlCl₃), the medium dose group (400 μM AlCl₃), the high dose group (800 μM AlCl₃), empty plasmid group, ApoEε4 group and 400 μM AlCl₃+ApoEε4 group. The cell viability was determined by CCK-8 kit after transfection for 48 h.The contents of total tau proteins, tau-181, tau-231, tau-262, tau-396 and Aβ42, were determined by ELISA kit. The interaction between AlCl₃ and ApoEε4 was analyzed by factorial design.ResultsWith the increase of aluminum exposure, SH-SY5Y cell viability decreased, and the expression of the total tau, tau-181, tau-231, tau-262, tau-396 and Aβ content increased. The viability of cells transfected with ApoEε4 is significantly lower than control group, and the expressions of total tau, tau-181, tau-231, tau-262, tau-396 and Aβ in ApoEε4 transfected cells were significantly higher than control group. The viability of cells treated with AlCl₃ plus ApoEε4 was lower than those treated with, either AlCl₃, or ApoEε4. The expression of total tau, tau-181, tau-231, tau-262, tau-396 and Aβ in the cells treated with AlCl₃ plus ApoEε4 were significantly higher than those in other groups (p < 0.05). Moreover, analyzing data based on the factorial design, there was existed an interaction between AlCl₃ and ApoEε4 (p < 0.05).ConclusionAl and ApoEε4 gene can cause morphological changes of SH-SY5Y cells, reduce cell activity, and have obvious cytotoxic effects, and increase the phosphorylation levels of tau and the deposition of Aβ increases. In the presence of both Al and ApoEε4 genes, the two factors interact with each other and show a synergistic effect.     

Ginsenoside Rd Attenuates Tau Protein Phosphorylation Via the PI3K/AKT/GSK-3β Pathway After Transient Forebrain Ischemia

Phosphorylated tau was found to be regulated after cerebral ischemia and linked to high risk for the development of post-stroke dementia. Our previous study showed that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, decreased tau phosphorylation in Alzheimer model. As an extending study, here we investigated whether Rd could reduce tau phosphorylation and sequential cognition impairment after ischemic stroke. Sprague–Dawley rats were subjected to focal cerebral ischemia. The tau phosphorylation of rat brains were analyzed following ischemia by Western blot and animal cognitive functions were examined by Morris water maze and Novel object recognition task. Ischemic insults increased the levels of phosphorylated tau protein at Ser199/202 and PHF-1 sites and caused animal memory deficits. Rd treatment attenuated ischemia-induced enhancement of tau phosphorylation and ameliorated behavior impairment. Furthermore, we revealed that Rd inhibited the activity of Glycogen synthase kinase-3β (GSK-3β), the most important kinase involving tau phosphorylation, but enhanced the activity of protein kinase B (PKB/AKT), a key kinase suppressing GSK-3β activity. Moreover, we found that LY294002, an antagonist for phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, abolished the inhibitory effect of Rd on GSK-3β activity and tau phosphorylation. Taken together, our findings provide the first evidence that Rd may reduce cerebral ischemia-induced tau phosphorylation via the PI3K/AKT/GSK-3β pathway.     

GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes

Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.