GSK3β Activity Research Articles

GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.

Hypoxia-induced lncRNA STEAP3-AS1 activates Wnt/β-catenin signaling to promote colorectal cancer progression by preventing m6A-mediated degradation of STEAP3 mRNA

BackgroundHypoxia, a typical hallmark of solid tumors, exhibits an essential role in the progression of colorectal cancer (CRC), in which the dysregulation of long non-coding RNAs (lncRNAs) is frequently observed. However, the underlying mechanisms are not clearly defined.MethodsThe TCGA database was analyzed to identify differential lncRNA expression involved in hypoxia-induced CRC progression. qRT-PCR was conducted to validate the upregulation of lncRNA STEAP3-AS1 in CRC cell lines and tumor-bearing mouse and zebrafish models under hypoxia. ChIP-qRT-PCR was used to detect the transcriptional activation of STEAP3-AS1 mediated by HIF-1α. RNA-seq, fluorescent in situ hybridization, RNA pulldown, RNA immunoprecipitation, co-immunoprecipitation, immunofluorescence and immunoblot experiments were used to ascertain the involved mechanisms. Functional assays were performed in both in vitro and in vivo models to investigate the regulatory role of STEAP3-AS1/STEAP3/Wnt/β-catenin axis in CRC proliferation and metastasis.ResultsHere, we identified a hypoxia-induced antisense lncRNA STEAP3-AS1 that was highly expressed in clinical CRC tissues and positively correlated with poor prognosis of CRC patients. Upregulation of lncRNA STEAP3-AS1, which was induced by HIF-1α-mediated transcriptional activation, facilitated the proliferation and metastasis of CRC cells both in vitro and in vivo. Mechanistically, STEAP3-AS1 interacted competitively with the YTH domain-containing family protein 2 (YTHDF2), a N6-methyladenosine (m6A) reader, leading to the disassociation of YTHDF2 with STEAP3 mRNA. This effect protected STEAP3 mRNA from m6A-mediated degradation, enabling the high expression of STEAP3 protein and subsequent production of cellular ferrous iron (Fe2+). Increased Fe2+ levels elevated Ser 9 phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and inhibited its kinase activity, thus releasing β-catenin for nuclear translocation and subsequent activation of Wnt signaling to support CRC progression.ConclusionsTaken together, our study highlights the mechanisms of lncRNA STEAP3-AS1 in facilitating CRC progression involving the STEAP3-AS1/STEAP3/Wnt/β-catenin axis, which may provide novel diagnostic biomarkers or therapeutic targets to benefit CRC treatment.Graphical abstractHypoxia-induced HIF-1α transcriptionally upregulates the expression of lncRNA STEAP3-AS1, which interacts competitively with YTHDF2, thus upregulating mRNA stability of STEAP3 and consequent STEAP3 protein expression. The enhanced STEAP3 expression results in production of cellular ferrous iron (Fe2+), which induces the Ser 9 phosphorylation and inactivation of GSK3β, releasing β-catenin for nuclear translocation and contributing to subsequent activation of Wnt signaling to promote CRC progression.

The Function of N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Negative Regulator in Tumor Cell Metastasis.

N-myc downstream-regulated gene 2 (NDRG2) is a tumor-suppressor gene that suppresses tumorigenesis and metastasis of tumors and increases sensitivity to anti-cancer drugs. In this review, we summarize information on the clinicopathological characteristics of tumor patients according to NDRG2 expression in various tumor tissues and provide information on the metastasis inhibition-related cell signaling modulation by NDRG2. Loss of NDRG2 expression is a prognostic factor that correlates with TNM grade and tumor metastasis and has an inverse relationship with patient survival in various tumor patients. NDRG2 inhibits cell signaling, such as AKT-, NF-κB-, STAT3-, and TGF-β-mediated signaling, to induce tumor metastasis, and induces activation of GSK-3β which has anti-tumor effects. Although NDRG2 operates as an adaptor protein to mediate the interaction between kinases and phosphatases, which is essential in regulating cell signaling related to tumor metastasis, the molecular mechanism of NDRG2 as an adapter protein does not seem to be fully elucidated. This review aims to assist the research design regarding NDRG2 function as an adaptor protein and suggests NDRG2 as a molecular target to inhibit tumor metastasis and improve the prognosis in tumor patients.

REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice.

Chronic hyperglycemia contributes to development of diabetic kidney disease by promoting glomerular injury. In this study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein regulated in development and DNA damage response 1 (REDD1) in the kidney in a manner that contributes to the development of oxidative stress and renal injury. After 16 weeks of streptozotocin-induced diabetes, albuminuria and renal hypertrophy were observed in wild-type (WT) mice coincident with increased renal REDD1 expression. In contrast, diabetic REDD1 knockout (KO) mice did not exhibit impaired renal physiology. Histopathologic examination revealed that glomerular damage including mesangial expansion, matrix deposition, and podocytopenia in the kidneys of diabetic WT mice was reduced or absent in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions enhanced REDD1 expression, increased reactive oxygen species (ROS) levels, and promoted cell death. In both the kidney of diabetic mice and in podocyte cultures exposed to hyperglycemic conditions, REDD1 deletion reduced ROS and prevented podocyte loss. Benefits of REDD1 deletion were recapitulated by pharmacological GSK3β suppression, supporting a role for REDD1-dependent GSK3β activation in diabetes-induced oxidative stress and renal defects. The results support a role for REDD1 in diabetes-induced renal complications.

GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia.

Apoptotic cell death is a deleterious consequence of hypoxia-induced cellular stress. The master hypoxamiR, microRNA-210 (miR-210), is considered the primary driver of the cellular response to hypoxia stress. We have recently demonstrated that miR-210 attenuates hypoxia-induced apoptotic cell death. In this paper, we unveil that the miR-210-induced inhibition of the serine/threonine kinase Glycogen Synthase Kinase 3 beta (GSK3β) in AC-16 cardiomyocytes subjected to hypoxia stress underlies the salutary protective response of miR-210 in mitigating the hypoxia-induced apoptotic cell death. Using transient overexpression vectors to augment miR-210 expression concomitant with the ectopic expression of the constitutive active GSK3β S9A mutant (ca-GSK3β S9A), we exhaustively performed biochemical and molecular assays to determine the status of the hypoxia-induced intrinsic apoptosis cascade. Caspase-3 activity analysis coupled with DNA fragmentation assays cogently demonstrate that the inhibition of GSK3β kinase activity underlies the miR-210-induced attenuation in the hypoxia-driven apoptotic cell death. Further elucidation and delineation of the upstream cellular events unveiled an indispensable role of the inhibition of GSK3β kinase activity in mediating the miR-210-induced mitigation of the hypoxia-driven BAX and BAK insertion into the outer mitochondria membrane (OMM) and the ensuing Cytochrome C release into the cytosol. Our study is the first to unveil that the inhibition of GSK3β kinase activity is indispensable in mediating the miR-210-orchestrated protective cellular response to hypoxia-induced apoptotic cell death.

Inhibition of NLRP1-Dependent Pyroptosis Prevents Glycogen Synthase Kinase-3β Overactivation-Induced Hyperphosphorylated Tau in Rats.

Our previous study indicated that inhibition of NLRP1-dependent pyroptosis could decrease intracerebroventricular (ICV) injection of a protein kinase A (PKA) agonist- or streptozotocin (STZ)-induced hyperphosphorylated tau. In this study, we used a glycogen synthase kinase-3β (GSK-3β) overactivation rat model to reconfirm our previous results. ICV injection of wortmannin (WT, a PI3K inhibitor) and GF-109203X (GFX, a PKC inhibitor) was used to induce overactivation of GSK-3β in rats. We injected NLRP1 siRNA together with WT/GFX to evaluate the effect of the inhibition of NLRP1-dependent neuronal pyroptosis on hyperphosphorylated tau. Our results indicated that ICV injection of NLRP1 siRNA prevented ICV-WT/GFX-induced neuronal death, further improving the spatial memory of the rats in the Morris water maze test. ICV injection of NLRP1 siRNA downregulated the expression of ASC, caspase-1, and GSDMD and the contents of IL-1β and IL-18 in rat brains. ICV injection of NLRP1 siRNA also decreased hyperphosphorylated tau and the activity of GSK-3β. Thus, these results support our previous study that NLRP1-dependent pyroptosis could enhance hyperphosphorylation of tau protein.

Total astragalosides promote oligodendrocyte precursor cell differentiation and enhance remyelination in cuprizone-induced mice through suppression of Wnt/β-catenin signaling pathway

Ethnopharmacological relevanceRadix Astragali is a traditional Chinese medicine with various pharmacological effects. Total astragalosides (TA), the main effective ingredients in Radix Astragali, exert properties including anti-oxidative stress, anti-neuroinflammation, and neuroprotection. We previously found that TA alleviated experimental autoimmune encephalomyelitis (EAE) progression, a widely used animal model of multiple sclerosis (MS). As a chronic demyelination disease, MS generally manifests myelin loss and fails to myelin regeneration. Regulation of oligodendrocyte progenitor cells (OPCs) differentiation and remyelination is the fundamental strategy for MS treatment. However, whether TA could directly promote OPCs differentiation and remyelination is still unknown. Aims of the studyThis study was aimed to investigate pro-differentiation and myelin regeneration effects of TA on OPCs and Cuprizone (CPZ)-induced demyelination mice, an animal model of MS, and to explore mechanism underlying from regulation of OPCs differentiation and maturation. Materials and methodsMice were orally given CPZ (400 mg/kg) daily for 4 weeks to induce myelin loss, and then treated with TA (25 and 50 mg/kg) daily for 1 week. Cell proliferation assay, Western blot, RT-PCR, immunocytochemistry and immunohistochemistry were performed to explore the mechanisms. The role of TA in oligodendrocyte differentiation and maturation was evaluated using MO3.13, a human oligodendrocytic hybrid cell line. ResultsTA was shown to mitigate behavioral impairment in CPZ-induced mice. It markedly ameliorated myelin loss and enhanced remyelination in the corpus callosum of mice, evidenced by increased expression of myelin basic protein (MBP) and the number of CC1+ newly generated oligodendrocytes (OLs). TA also enhanced the expression of MBP at both mRNA and protein levels in MO3.13 cells. In CPZ-induced mice and MO3.13 cells, TA remarkably promoted the activation of GSK3β, repressed the phosphorylation of β-catenin, reduced the expression of transcription factor 4 and inhibitor of DNA binding 2. The agonist of β-catenin, SKL2001, partially abolished the pro-differentiation effect of TA in MO3.13 cells. ConclusionsTaken together, we clarified that TA could effectively enhance the differentiation and maturation of OPCs and accelerate remyelination in CPZ-induced mice through inhibition of Wnt/β-catenin signaling pathway. This study provides new insight into the beneficial effect of TA in the treatment of MS.

Vitamin C deficiency induces hypoglycemia and cognitive disorder through S-nitrosylation-mediated activation of glycogen synthase kinase 3β

Vitamin C (VC, l-ascorbic acid) is an essential nutrient that plays a key role in metabolism and functions as a potent antioxidant in regulating the S-nitrosylation and denitrosylation of target proteins. The precise function of VC deprivation in glucose homeostasis is still unknown. In the absence of L-gulono-1,4-lactone oxidoreductase, an essential enzyme for the last step of VC synthesis, VC deprivation resulted in persistent hypoglycemia and subsequent impairment of cognitive functions in female but not male mouse pups. The cognitive disorders caused by VC deprivation were largely reversed when these female pups were given glucose. VC deprivation-induced S-nitrosylation of glycogen synthase kinase 3β (GSK3β) at Cys14, which activated GSK3β and inactivated glycogen synthase to decrease glycogen synthesis and storage under the feeding condition, while VC deprivation inactivated glycogen phosphorylase to decrease glycogenolysis under the fasting condition, ultimately leading to hypoglycemia and cognitive disorders. Treatment with Nω-Nitro-l-arginine methyl ester (l-NAME), a specific inhibitor of nitric oxide synthase, on the other hand, effectively prevented S-nitrosylation and activation of GSK3β in female pups in response to the VC deprivation and reversed hypoglycemia and cognitive disorders. Overall, this research identifies S-nitrosylation of GSK3β and subsequent GSK3β activation as a previously unknown mechanism controlling glucose homeostasis in female pups in response to VC deprivation, implying that VC supplementation in the prevention of hypoglycemia and cognitive disorders should be considered in the certain groups of people, particularly young females.

Anti-Inflammatory Effects of Auranamide and Patriscabratine-Mechanisms and In Silico Studies.

Auranamide and patriscabratine are amides from Melastoma malabathricum (L.) Smith. Their anti-inflammatory activity and nuclear factor erythroid 2-related factor 2 (NRF2) activation ability were evaluated using Escherichia coli lipopolysaccharide (LPSEc)-stimulated murine macrophages (RAW264.7) and murine hepatoma (Hepa-1c1c7) cells, respectively. The cytotoxicity of the compounds was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The anti-inflammatory activity was determined by measuring the nitric oxide (NO) production and pro-inflammatory cytokines (Interleukin (IL)-1β, Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-6) and mediators (NF-κB and COX-2). NRF2 activation was determined by measuring the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) quinone oxidoreductase 1 (NQO1), nuclear NRF2 and hemeoxygenase (HO)-1. In vitro metabolic stability was assessed using the mouse, rat, and human liver microsomes. The compounds were non-toxic to the cells at 10 μM. Both compounds showed dose-dependent effects in downregulating NO production and pro-inflammatory cytokines and mediators. The compounds also showed upregulation of NQO1 activity and nuclear NRF2 and HO-1 levels. The compounds were metabolically stable in mouse, rat and human liver microsomes. The possible molecular targets of NRF2 activation by these two compounds were predicted using molecular docking studies and it was found that the compounds might inhibit the Kelch domain of KEAP1 and GSK-3β activity. The physicochemical and drug-like properties of the test compounds were predicted using Schrodinger small molecule drug discovery suite (v.2022-2).

Abstract P1078: Early Trypanosoma Cruzi Infection Of Heart Endothelial Cells Modulates Hippo And Wnt Signaling Pathways In TSP-1 Dependent Manner

Trypanosoma cruzi , an obligate intracellular protozoan parasite, is the etiological agent of Chagas Disease. Due to globalization, the disease is now present in all economically advanced countries of the world. About 40% of T. cruzi infected individuals will develop cardiac, neurological, and/or gastrointestinal pathology, with Chagasic Cardiomyopathy being the most prevalent pathology. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC), especially heart endothelium is currently unknown. Previous studies in our lab showed that the parasite dysregulates the expression of host genes, such as Thrombospondin-1 (TSP-1), to facilitate infection. Hippo and Wnt signaling pathways, in particular, have been implicated in cardiac development and disease pathogenesis. Here, we aim to evaluate the significance of TSP-1 in Hippo and Wnt signaling during the course of early T. cruzi infection. We challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling by immunoblotting, YAP/β-catenin crosstalk via immunofluorescence assays, and parasite infectivity using β-catenin inhibitor. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a and enhanced the activation of GSK-3β simultaneously. In WT MHEC, the levels of Wnt-5a, p-GSK-3β were decreased compared to uninfected control. We also observed a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC. Interestingly in WT MHEC, nuclear colocalization of β-catenin/YAP remained steady and reduced during later parasite challenge. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor-treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that TSP1 plays an important role in regulating Wnt/Hippo crosstalk and may be important potential therapeutic targets.

Glycogen Synthase Kinase 3β Involvement in Neuroinflammation and Neurodegenerative Diseases.

GSK-3β activity has been strictly related to neuroinflammation and neurodegeneration. Alzheimer's disease is the most studied neurodegenerative disease, but GSK-3β seems to be involved in almost all neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, and the autoimmune disease multiple sclerosis. This review aims to help researchers both working on this research topic or not to have a comprehensive overview of GSK-3β in the context of neuroinflammation and neurodegeneration. Literature has been searched using PubMed and SciFinder databases by inserting specific keywords. A total of more than 500 articles have been discussed. First of all, the structure and regulation of the kinase were briefly discussed, and then, specific GSK-3β implications in neuroinflammation and neurodegenerative diseases were illustrated with the help of figures, to conclude with a comprehensive overview on the most important GSK-3β and multitarget inhibitors. The structure and IC50 values at the target kinase have been reported for all the discussed compounds. GSK-3β is involved in several signaling pathways in neurons, glial cells and immune cells. The fine regulation and interconnection of all these pathways are at the base of the rationale use of GSK-β inhibitors in neuroinflammation and neurodegeneration. Some compounds are now under clinical trials. Despite this, the compounds' pharmacodynamic and ADME/Tox profiles were often not fully characterized which is deleterious in such a complex system.

Compressive force regulates GSK-3β in osteoclasts contributing to alveolar bone resorption during orthodontic tooth movement in vivo

BackgroundOrthodontic tooth movement mainly depends on biological and mechanical reactions in the periodontium, such as the indispensable reconstruction process of the periodontal ligament and alveolar bone. To explore whether orthodontic compressive force can induce bone resorption during orthodontic tooth movement by regulating the GSK-3β/β-catenin pathway. MethodsWe established orthodontic tooth movement models in Sprague-Dawley rats. In addition, compressive force-induced bone resorption that occurred during orthodontic tooth movement was analyzed by HE staining and micro-CT. The number and distribution of osteoclasts were observed by TRAP staining. Furthermore, pressure-induced bone resorption mediated by the GSK-3β/β-catenin signaling pathway was analyzed by immunohistochemistry. ResultsAs shown by the micro-CT results, bone parameters, such as bone mineral density (BMD), the bone volume fraction (BV/TV), and trabecular thickness (Tb. Th), were significantly decreased under orthodontic compressive force stimulation, in contrast with the dramatically increased trabecular spacing (Tb. Sp). During the process of tooth movement, the compressive force can induce bone resorption on the side with the force, which increases the expression of phosphorylated Ser-GSK-3β and activation of the β-catenin signaling pathway. Additionally, downregulation of the GSK-3β activity further caused the downregulation of bone parameters, leading to bone loss. The TRAP staining and immunohistochemistry staining results indicated that orthodontic compressive force influenced osteoclast formation and the secretion of osteoclast-related cytokines, matrix metallopeptidase 9 (MMP-9) and receptor activator of nuclear factor-κB ligands (RANKLs), which is also related to the duration of orthodontic force. ConclusionsThese results indicated that the GSK-3β inhibitor can promote osteoclast formation on the side with orthodontic compressive force. In addition, the activation of the GSK-3β/β-catenin signaling pathway contributes to bone reconstruction caused by orthodontic compressive force. Therefore, the GSK-3β/β-catenin signaling pathway can be a potential target for further clinical applications.

Furanoditerpenes from Spongia (Spongia) tubulifera Display Mitochondrial-Mediated Neuroprotective Effects by Targeting Cyclophilin D.

Neuroprotective properties of five previously described furanoditerpenes 1-5, isolated from Spongia (Spongia) tubulifera, were evaluated in an in vitro oxidative stress model in SH-SY5Y cells. Dose-response treatments revealed that 1-5 improved cell survival at nanomolar concentrations through the restoration of mitochondrial membrane potential and the reduction of reactive oxygen species. Their ability to prevent the mitochondrial permeability transition pore opening was also assessed, finding that 4 and 5 inhibited the channel at 0.001 μM. This inhibition was accompanied by a decrease in the expression of cyclophilin D, the main regulator of the pore, which was also reduced by 1 and 2. However, the activation of ERK and GSK3β, upstream modulators of the channel, was not affected by compounds. Therefore, their ability to bind cyclophilin D was evaluated by surface plasmon resonance, observing that 2-5 presented equilibrium dissociation constants in the micromolar range. All compounds also showed affinity for cyclophilin A, being 1 selective toward this isoform, while 2 and 5 exhibited selectivity for cyclophilin D. When the effects on the intracellular expression of cyclophilins A-C were determined, it was found that only 1 decreased cyclophilin A, while cyclophilins B and C were diminished by most compounds, displaying enhanced effects under oxidative stress conditions. Results indicate that furanoditerpenes 1-5 have mitochondrial-mediated neuroprotective properties through direct interaction with cyclophilin D. Due to the important role of this protein in oxidative stress and inflammation, compounds are promising drugs for new therapeutic strategies against neurodegeneration.

Cdc42 Promotes Axonogenesis of Primary Hippocampal Neurons by Inhibiting Glycogen Synthase Kinase-3β.

Progressive axon degeneration is a common pathological feature of neurodegenerative diseases. Cdc42 is a member of the Rho GTPase family that participates in axonogenesis. GSK-3β is a serine/threonine kinase highly implicated in neuronal development and neurodegeneration. This study aimed to examine whether cdc42 promotes axonogenesis by regulating GSK-3β activity. Hippocampal neurons were isolated from neonatal Sprague-Dawley rats and transfected with designated plasmid vectors to alter the activities of cdc42 and GSK-3β. LiCl treatment was used to inhibit the GSK-3β activity in primary neurons. GSK-3β activity was determined by an enzyme activity assay kit. Immunofluorescence staining was used to detect axons stained with anti-Tau-1 antibody and dendrites stained with anti-MAP2 antibody. Transfection with an active cdc42 mutant (cdc42F28L) decreased the activity of GSK-3β and induced axonogenesis in primary rat hippocampal neurons, while transfection with a negative cdc42 mutant (cdc42N17) resulted an opposite effect. Moreover, transfection with plasmid vectors carrying wild-type GSK-3β or a constitutively active GSK3β mutant (GSK-3β S9A) increased the activity of GSK-3β and attenuated axonogenesis of primary hippocampal neurons with excessive cdc42 activity, whereas inhibition of GSK-3β by LiCl abolished the inhibitory effect of the negative cdc42 mutant on axonogenesis. This study suggests that cdc42 induces axonogenesis of primary rat hippocampal neurons via inhibiting GSK-3β activity. These findings support further investigation into the mechanisms of cdc42/GSK-3β-mediated axonogenesis.

Isolation and characterization of neurotoxic astrocytes derived from adult triple transgenic Alzheimer's disease mice

Alzheimer's disease has been considered mostly as a neuronal pathology, although increasing evidence suggests that glial cells might play a key role in the disease onset and progression. In this sense, astrocytes, with their central role in neuronal metabolism and function, are of great interest for increasing our understanding of the disease. Thus, exploring the morphological and functional changes suffered by astrocytes along the course of this disorder has great therapeutic and diagnostic potential.In this work we isolated and cultivated astrocytes from symptomatic 9-10-months-old adult 3xTg-AD mice, with the aim of characterizing their phenotype and exploring their pathogenic potential. These “old” astrocytes occurring in the 3xTg-AD mouse model of Alzheimer's Disease presented high proliferation rate and differential expression of astrocytic markers compared with controls. They were neurotoxic to primary neuronal cultures both, in neuronal-astrocyte co-cultures and when their conditioned media (ACM) was added into neuronal cultures. ACM caused neuronal GSK3β activation, changes in cytochrome c pattern, and increased caspase 3 activity, suggesting intrinsic apoptotic pathway activation. Exposure of neurons to ACM caused different subcellular responses. ACM application to the somato-dendritic domain in compartmentalised microfluidic chambers caused degeneration both locally in soma/dendrites and distally in axons. However, exposure of axons to ACM did not affect somato-dendritic nor axonal integrity.We propose that this newly described old 3xTg-AD neurotoxic astrocytic population can contribute towards the mechanistic understanding of the disease and shed light on new therapeutical opportunities.

Calcium controlled NFATc1 activation enhances suppressive capacity of regulatory T cells isolated from generalized vitiligo patients.

NFATs and FOXP3 are linked with impaired regulatory T-cells (Tregs) in generalized vitiligo (GV). To elucidate calcium mediated NFATc1 signalling pathway and its effect on Treg suppressive capacity in GV. Calcium levels, calcineurin, NFATc1 and GSK-3β activity and cell proliferation were assessed in 52 GV patients and 50 controls by calcium assay kit, calcineurin phosphatase assay kit, TransAM NFATc1 kit, GSK-3β ELISA and BrdU cell proliferation assay. Transcripts (CNB, CAM, GSK3B, DYRK1A and calcium channel genes) and protein (IFN-γ, IL-10 and TGF-β) expressions were assessed by qPCR and ELISA, respectively. Reduced plasma and intracellular Tregs calcium levels and ORAI1 transcripts suggested altered calcium homeostasis in GV Tregs (p=0.00387, p=0.0048, p < 0.0001), which led to decreased calcineurin and NFATc1 activity in GV Tregs (p=0.0299, p < 0.0001). CNB and CAM transcripts were reduced in GV Tregs (p < 0.0001, p=0.0004). GSK-3β activity, GSK3B and DYRK1A transcripts significantly increased in GV Tregs (p=0.0134, p < 0.0001 and p < 0.0001). Plasma (p=0.0225, p=0.032) and intracellular Treg (p=0.0035, p=0.005) calcium levels, calcineurin (p=0.001) and NFATc1 (p=0.001, p < 0.0001) activity and ORAI1 (p=0.0093, p < 0.0001), CAM and CNB (p=0.0214) transcripts significantly decreased in active vitiligo (AV) and severe GV (sGV) Tregs. Calcium treatment significantly increased intracellular calcium and ORAI1 transcripts in GV Tregs (p=0.0042, p=0.0035). Moreover, calcium treatment enhanced calcineurin and NFATc1 activity in GV Tregs (p=0.0128, p < 0.0001). Remarkably, calcium treatment increased Treg mediated suppression of CD4+ and CD8+ T-cells (p=0.015, p=0.006) in GV and increased Tregs associated cytokines: IL-10 (p=0.0323, p=0.009), TGF-β (p=0.0321, p=0.01) and decreased IFN-γ production (p=0.001, p=0.016) by CD4+ and CD8+ T-cells. Intracellular calcium levels positively correlated with calcineurin (r=0.83; p < 0.0001) and NFATc1 (r=0.61; p < 0.0001) activity, suggesting the enhanced Treg immunosuppressive capacity after calcium treatment. Our study for the first time suggests that reduced plasma calcium and ORAI1 transcripts are linked to calcium uptake defects in Tregs, which leads to reduced calcineurin and NFATc1 activation, thereby contributing to decreased Tregs immunosuppressive capacity in GV. Elevated GSK-3β activity and GSKB and DYRK1A transcripts are involved in reduced NFATc1 activity in GV Tregs. Overall, the study suggests that calcium-NFATc1-signalling pathway is likely to be involved in defective Tregs function and can be implicated for development of effective Treg mediated therapeutics for GV.

A network pharmacology-based study on the mechanism of astragaloside IV alleviating renal fibrosis through the AKT1/GSK-3β pathway

Ethnopharmacological revelvanceAstragaloside IV, a glycoside derived from Astragalus membranaceus, has anti-renal fibrosis effects. However, its mechanism of action has not yet been fully elucidated. Aim of the studyThe purpose of this study was to investigate the anti-fibrotic effect of AS-IV and to clarify its underlying mechanism. Materials and methodsThe network pharmacology method, molecular docking and surface plasmon resonance (SPR) was used to identify potential targets and pathways of AS-IV. A unilateral ischemia-reperfusion injury (UIRI) animal model, as well as TGF-β1-induced rat renal tubular epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were used to investigate and validate the anti-fibrotic activity and pharmacological mechanism of AS-IV. Network pharmacology was performed to construct a drug-target-pathway network. The anti-fibrosis effect of AS-IV was determined using hematoxylin-eosin (H&E) and MASSON staining, as well as immunostaining methods. qRT-PCR and western blotting were used to elucidate and validate the mechanism of AS-IV. ResultsNetwork pharmacology revealed that the PI3K/AKT pathway is an important pathway in AS-IV. AS-IV inhibited the expression of α-SMA, collagen I, and fibronectin in NRK-52E, NRK-49F, and UIRI rats, and reduced serum creatinine and blood urea nitrogen levels in UIRI rats. AS-IV inhibited AKT phosphorylation, blocked GSK-3β phosphorylation, and restored GSK-3β activity, which contributed to the degradation of β-catenin, thereby preventing epithelial-mesenchymal transition (EMT). ConclusionAstragaloside IV alleviated renal fibrosis through the AKT1/GSK-3β pathway. In addition, our findings indicate that the network pharmacology method is a powerful tool for exploring the pharmacological mechanisms of drugs.

High-throughput screening identifies stevioside as a potent agent to induce apoptosis in bladder cancer cells

BackgroundBladder cancer (BC) is a global health issue that lacks effective treatment strategies. Growing evidence suggests that various natural products possess anti-tumour effects. This study aims to identify a novel agent that can be used in the treatment of BC. MethodsHigh-throughput screening was conducted to search for potential anti-BC natural agents. Cell viabilities were measured by the CCK-8 assay. Cell death, cellular reactive oxygen species (ROS), and mitochondrial outer membrane potential (MOMP) were measured by flow cytometry. RNA sequencing was conducted to identify the affected signalling pathways. Western blots were used to measure the change of proteins. Xenografts models were used to assess the anti-tumour effects in vivo. ResultsThrough high-throughput screening, we identified stevioside, a diterpenoid glycoside isolated from Stevia rebaudiana, which selectively inhibited the viability of BC cells and induced their intrinsic apoptosis sparing normal cells. Stevioside also induced mitochondrial stress in BC cells, and activated Bax by downregulating Mcl-1 and upregulating Noxa. RNA sequencing revealed that stevioside treatment caused activation of GSK-3β and endoplasmic reticulum (ER) stress signalling pathways. Activation of GSK-3β induced upregulation of FBXW7, which effectuated the downregulation of Mcl-1. In addition, activation of GSK-3β triggered ER stress, leading to the upregulation of Noxa. Further investigations revealed that the accumulation of ROS was responsible for the activation of the GSK-3β signalling pathway in BC cells. Moreover, we also found that stevioside inhibited the growth of BC cells in vivo. ConclusionsCollectively, our data suggest that stevioside can be a potential agent for the treatment of BC.

Phosphodiesterase 5 inhibitor mirodenafil ameliorates Alzheimer-like pathology and symptoms by multimodal actions

BackgroundAlzheimer’s disease (AD) pathology is associated with complex interactions among multiple factors, involving an intertwined network of various signaling pathways. The polypharmacological approach is an emerging therapeutic strategy that has been proposed to overcome the multifactorial nature of AD by targeting multiple pathophysiological factors including amyloid-β (Aβ) and phosphorylated tau. We evaluated a blood-brain barrier penetrating phosphodiesterase 5 (PDE5) inhibitor, mirodenafil (5-ethyl-2-7-n-propyl-3,5-dihydrro-4H-pyrrolo[3,2-d]pyrimidin-4-one), for its therapeutic effects on AD with polypharmacological properties.MethodsTo evaluate the potential of mirodenafil as a disease-modifying AD agent, mirodenafil was administered to test its effects on the cognitive behaviors of the APP-C105 AD mouse model using the Morris water maze and passive avoidance tests. To investigate the mechanisms of action that underlie the beneficial disease-modifying effects of mirodenafil, human neuroblastoma SH-SY5Y cells and mouse hippocampal HT-22 cells were used to show mirodenafil-induced alterations associated with the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG)/cAMP-responsive element-binding protein (CREB) pathway, apoptotic cell death, tau phosphorylation, amyloidogenesis, the autophagy-lysosome pathway, glucocorticoid receptor (GR) transcriptional activity, and the Wnt/β-catenin signaling.ResultsHere, mirodenafil is demonstrated to improve cognitive behavior in the APP-C105 mouse model. Mirodenafil not only reduced the Aβ and phosphorylated tau burdens in vivo, but also ameliorated AD pathology induced by Aβ through the modulation of the cGMP/PKG/CREB signaling pathway, glycogen synthase kinase 3β (GSK-3β) activity, GR transcriptional activity, and the Wnt/β-catenin signaling in neuronal cells. Interestingly, homodimerization and nuclear localization of GR were inhibited by mirodenafil, but not by other PDE5 inhibitors. In addition, only mirodenafil reduced the expression levels of the Wnt antagonist Dickkopf-1 (Dkk-1), thus activating the Wnt/β-catenin signaling.ConclusionsThese findings strongly suggest that the PDE5 inhibitor mirodenafil shows promise as a potential polypharmacological drug candidate for AD treatment, acting on multiple key signaling pathways involved in amyloid deposition, phosphorylated tau burden, the cGMP/PKG/CREB pathway, GSK-3β kinase activity, GR signaling, and the Wnt/β-catenin signaling. Mirodenafil administration to the APP-C105 AD mouse model also improved cognitive behavior, demonstrating the potential of mirodenafil as a polypharmacological AD therapeutic agent.

Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity.

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogen-activated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.